Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity

ABSTRACT

The present invention relates to the field glycosylation engineering of proteins. More particular, the present invention is directed to the glycosylation engineering of proteins to provide proteins with improved therapeutic properties, e.g., antibodies, antibody fragments, or a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin, with enhanced Fc-mediated cellular cytotoxicity.

I. RELATION TO OTHER APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/294,548, filed Apr. 20, 1999 (U.S. Pat. No. 6,602,684), which claimsthe benefit of United States Provisional Application Ser. No.60/082,581, filed Apr. 20, 1998, both of which are incorporated hereinby reference in their entirety.

II. FIELD OF THE INVENTION

The present invention relates to the field of glycosylation engineeringof proteins. More particularly, the present invention relates toglycosylation engineering to generate proteins with improved therapeuticproperties, including antibodies with enhanced antibody-dependentcellular cytotoxicity.

III. BACKGROUND OF THE INVENTION

Glycoproteins mediate many essential functions in human beings, othereukaryotic organisms, and some prokaryotes, including catalysis,signalling, cell-cell communication, and molecular recognition andassociation. They make up the majority of non-cytosolic proteins ineukaryotic organisms. Lis and Sharon, 1993, Eur. J. Biochem. 218:1-27.Many glycoproteins have been exploited for therapeutic purposes, andduring the last two decades, recombinant versions ofnaturally-occurring, secreted glycoproteins have been a major product ofthe biotechnology industry. Examples include erythropoietin (EPO),therapeutic monoclonal antibodies (therapeutic mAbs), tissue plasminogenactivator (tPA), interferonβ, (IFNβ), granulocyte-macrophage colonystimulating factor (GM-CSF), and human chorionic gonadotrophin (hCH).Cumming et al., 1991, Glycobiology 1:115-130.

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. Jenkins et al, 1996,Nature Biotechn. 14:975-981.

Mammalian cells are the preferred hosts for production of therapeuticglycoproteins, due to their capability to glycosylate proteins in themost compatible form for human application. Cumming, 1991, supra;Jenkins et al., 1996, supra. Bacteria very rarely glycosylate proteins,and like other types of common hosts, such as yeasts, filamentous fungi,insect and plant cells, yield glycosylation patterns associated withrapid clearance from the blood stream, undesirable immune interactions,and in some specific cases, reduced biological activity. Among mammaliancells, Chinese hamster ovary (CHO) cells have been most commonly usedduring the last two decades. In addition to giving suitableglycosylation patterns, these cells allow consistent generation ofgenetically stable, highly productive clonal cell lines. They can becultured to high densities in simple bioreactors using serum-free media,and permit the development of safe and reproducible bioprocesses. Othercommonly used animal cells include baby hamster kidney (BHK) cells, NS0-and SP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. Jenkins et al., 1996, supra.

The glycosylation of recombinant therapeutic proteins produced in animalcells can be engineered by overexpression of glycosyl transferase genesin host cells. Bailey, 1991, Science 252:1668-1675. However, previouswork in this field has only used constitutive expression of theglycoprotein-modifying glycosyl transferase genes, and little attentionhas been paid to the expression level.

IV. SUMMARY OF THE INVENTION

The present invention is directed, generally, to host cells and methodsfor the generation of proteins having an altered glycosylation patternresulting in improved therapeutic values. In one specific embodiment,the invention is directed to host cells that have been engineered suchthat they are capable of expressing a preferred range of aglycoprotein-modifying glycosyl transferase activity which increasescomplex N-linked oligosaccharides carrying bisecting GlcNAc. In otherembodiments, the present invention is directed to methods for thegeneration of modified glycoforms of glycoproteins, for exampleantibodies, including whole antibody molecules, antibody fragments, orfusion proteins that include a region equivalent to the Fc region of animmunoglobulin, having an enhanced Fc-mediated cellular cytotoxicity,and glycoproteins so generated. The invention is based, in part, on theinventors'discovery that there is an optimal range ofglycoprotein-modifying glycosyl transferase expression for themaximization of complex N-linked oligosaccharides carrying bisectingGlcNAc.

More specifically, the present invention is directed to a method forproducing altered glycoforms of proteins having improved therapeuticvalues, e.g., an antibody which has an enhanced antibody dependentcellular cytotoxicity (ADCC), in a host cell. The invention provideshost cells which harbor a nucleic acid encoding the protein of interest,e.g., an antibody, and at least one nucleic acid encoding aglycoprotein-modifying glycosyl transferase. Further, the presentinvention provides methods and protocols of culturing such host cellsunder conditions which permit the expression of said protein ofinterest, e.g., the antibody having enhanced antibody dependent cellularcytotoxicity. Further, methods for isolating the so generated proteinhaving an altered glycosylation pattern, e.g., the antibody withenhanced antibody dependent cellular cytotoxicity, are described.

Furthermore, the present invention provides alternative glycoforms ofproteins having improved therapeutic properties. The proteins of theinvention include antibodies with an enhanced antibody-dependentcellular cytotoxicity (ADCC), which have been generated using thedisclosed methods and host cells.

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the representation of typical oligosaccharide structures.

FIG. 2 depicts a Western blot analysis of tetracycline-regulatedexpression of GnT III in two different tTA-producing CHO clones. CHOt2(lanes A and B) and CHOt17 (lanes C and D) cells were transfected withthe pUDH10-3GnTIIIm expression vector and cultured for 36 h in theabsence (lanes A and C) or presence of tetracycline, at a concentrationof 400 ng/ml (lanes B and D). Cell lysates were then prepared forwestern blot analysis probing with an antibody (9E10), which recognizesspecifically the c-myc tag added to GnT III at its carboxy-terminus.

FIG. 3 depicts determination of the range of tetracycline concentrationswhere myc-tagged GnT III expression can be controlled. CHOt17 cells weretransfected with the pUDH10-3-GnTIIIm expression vector and thencultured for 48 h in the presence of the indicated concentrations oftetracycline. GnT III levels in cell lysates from these cultures werecompared using western blot analysis. GnT III was detected via the c-myctag using 9E10 antibody.

FIGS. 4A through 4B depict screening of CHO clones for stable,tetracycline-regulated expression of GnT V (FIG. 4A) or myc-tagged GnTIII (FIG. 4B) glycosyltransferases by western blot analysis. CHOt17cells were co-transfected with a vector for expression of puromycinresistance (pPUR) and either pUHD10-3GnTV (FIG. 4A) or pUDH10-3GnTIIIm(FIG. 4B) and stable CHO clones were selected for resistance topuromycin (7.5 μ/ml), in the presence of tetracycline (2 μg/ml). Eightclones (1-8) for each glycosyltransferase were cultured for 48 h in theabsence or presence (+) of tetracycline (2 μg/ml) and analyzed bywestern blot using either an anti-GnT V antibody (FIG. 4A) or ananti-myc (9E10) antibody (FIG. 4B).

FIGS. 5A and 5B depict verification of activity of heterologous GnT V(FIG. 5A) and GnT III (FIG. 5B) glycosyltransferaseas in vivo by lectinblot analysis. Cellular glycoproteins from various stable clones(numbered as in FIG. 4), cultured in the absence or presence (+) oftetracycline (2 μg/ml), were resolved by SDS-PAGE, blotted to amembrane, and probed with either L-PHA (FIG. 5A) or E-PHA (FIG. 5B)lectins. These lectins bind with higher affinity to the oligosaccharideproducts of reactions catalyzed by GnT V and GnT III, respectively, thanto the oligosaccharide substrates of these reactions. A molecular weightmarker (MWM) was run in parallel. A comparison of lectin blots in FIGS.5A and 5B indicates a broader range of substrates, among the endogenousCHO cell glycoproteins, for GnT III (FIG. 5B) than for GnT V (FIG. 5A).

FIGS. 6A through 6D depict inhibition of cell growth uponglycosyltransferase overexpression. CHO-tet-GnTIIIm cells were seeded to5-10% confluency and cultured in the absence (FIGS. 6A and 6B) orpresence (FIGS. 6C and 6D) of tetracycline. Cultures were photographed45 (FIGS. 6A and 6C) and 85 (FIGS. 16B and 6D) hours after seeding.

FIG. 7 depicts sequences of oligonucleotide primers used in PCRs for theconstruction of the chCE7 heavy chain gene: CE7VHPCR1.fwd (SEQ ID NO:1),CE7VHPCR2.fwd (SEQ ID NO:2), CE7VHPCR(1+2).rev (SEQ ID NO:3),hGamma1CH1.fwd (SEQ ID NO:4), hGamma1CH1.rev (SEQ ID NO:5),hGamma1CH2.fwd (SEQ ID NO:6), hGamma1CH2.rev (SEQ ID NO:7),hGamma1CH3.fwd (SEQ ID NO:8), hGamma1CH3.rev (SEQ ID NO:9). Forward andreverse primers are identified by the suffixes “.fwd” and “.rev”,respectively. Overlaps between different primers, necessary to carry outsecondary PCR steps using the product of a primary PCR step as atemplate, are indicated. Restriction sites introduced, sequencesannealing to the CE7 chimeric genomic DNA, and they synthetic leadersequence introduced, are also indicated.

FIG. 8 depicts sequence of oligonucleotide primers used in PCRs for theconstruction of the chCE7 light chain gene: CE7VLPCR1.fwd (SEQ IDNO:10), CE7VLPCR2.fwd (SEQ ID NO:11), CE7VLPCR(1+2).rev (SEQ ID NO:12),hKappa.fwd (SEQ ID NO:13), hKappa.rev (SEQ ID NO:14). Forward andreverse primers are identified by the suffixes “.fwd” and “.rev”,respectively. Overlaps between different primers, necessary to carry outsecondary PCR steps using as a template the product of a primary PCRstep, are indicated. Restriction sites introduced, sequences annealingto the CE7 chimeric genomic DNA, and the leader sequence introduced, arealso indicated.

FIG. 9 depicts MALDI/TOF-MS spectra of neutral oligosaccharide mixturesfrom chCE7 samples produced either by SP2/0 mouse myeloma cells (FIG.9A, oligosaccharides from 50 μg of CE7-SP2/0), or by CHO-tetGnTIII-chCE7cell cultures differing in the concentration of tetracycline added tothe media, and therefore expressing the GnT III gene at differentlevels. In decreasing order of tetracycline concentration, i.e.,increasing levels of GnT III gene expression, the latter samples are:CE7-2000t (FIG. 9B, oligosaccharides from 37.5 μg of antibody), CE7-60t(FIG. 9C, oligosaccharides from 37.5 μg of antibody, CE7-30t (FIG. 9D,oligosaccharides from 25 μg of antibody) and CE7-15t (FIG. 9E,oligosaccharides from 10 μg of antibody).

FIG. 10 depicts N-linked oligosaccharide biosynthetic pathways leadingto bisected complex oligosaccharides via a GnT III-catalyzed reaction. Mstands for Mannose; Gn, N-acetylglucosamine (GlcNAc); G, galactose;Gn^(b), bisecting GlcNAc; f, fucose. The oligosaccharide nomenclatureconsists of enumerating the M, Gn, and G residues attached to the coreoligosaccharide and indicating the presence of a bisecting GlcNAc byincluding a Gn^(b). The oligosaccharide core is itself composed of 2 Gnresidues and may or may not include a fucose. The major classes ofoligosaccharides are shown inside dotted frames. Man I stands for Golgimannosidase; GnT, GlcNAc transferase; and GalT, forgalactosyltransferase. The mass associated with the major,sodium-associated oligosaccharide ion that is observed MALDI/TOF-MSanalysis is shown beside each oligosaccharide. For oligosaccharideswhich can potentially be core-fucosylated, the masses associated withboth fucosylated (+f) and non-fucosylated (−f) forms are shown.

FIG. 11 depicts N-linked oligosaccharide biosynthetic pathway leading tobisected complex and bisected hybrid oligosaccharides via GnTIII-catalyzed reactions. M stands for mannose; Gn N-acetylglucosamine(GlcNAc); G, galactose; Gn^(b), bisecting GlcNAc; f, fucose. Theoligosaccharide nomenclature consists of enumerating the M, Gn, and Gresidues attached to the common oligosaccharide and indicating thepresence of bisecting GlcNAc by including a Gn^(b). The oligosaccharidecore is itself composed of 2 Gn residues and may or not include afucose. The major classes of oligosaccharides are shown inside dottedframes. Man I stands for Golgi mannosidase; TnT, GlcNAc transferase; andGalT, for galactosyltransferase. The mass associated with major,sodium-associated oligosaccharide ion that is observed in MALDI/TOF-MSanalysis is shown beside each oligosaccharide. For oligosaccharideswhich can potentially be core-fucosylated, the masses associated withboth fucosylated (+f) and non-fucosylated (−f) forms are shown.

FIG. 12 depicts ADCC activity of different chCE7 samples. Lysis ofIMR-32 neuroblastoma cells by human lymphocytes (target:effector ratioof 1:19, 16 h incubation at 37° C.), mediated by differentconcentrations of chCE7 samples, was measured via retention of afluorescent dye. The percentage of cytotoxicity is calculated relativeto a total lysis control (by means of a detergent), after subtraction ofthe signal in the absence of antibody.

FIG. 13 depicts the GnT III expression of different cultures ofCHO-tet-GnTIII grown at different tetracycline concentrations used toproduce distinct C2B8 antibody samples. Cell lysates from each culturegrown at 2000 ng/ml (Lane C) and 25 ng/ml (Lane D) tetracyclineconcentrations were resolved by SDS-PAGE, blotted onto a membrane, andprobed with 9E10 (see supra) and anti-mouse horseradish peroxidase asprimary and secondary antibodies, respectively. Lane A depicts anegative control.

FIGS. 14A and 14B depict the specificity of antigen binding of the C2B8anti-CD20 monoclonal antibody using an indirect immunofluorescence assaywith cells in suspension. CD20 positive cells (SB cells; ATCC depositno. ATCC CCL120) and CD20 negative cells (HSB cells; ATCC deposit no.ATCC CCL120.1), FIGS. 14A and 14B respectively, were utilized. Cells ofeach type were incubated with C2B8 antibody produced at 25 ng/mltetracycline as a primary antibody. Negative controls included HBSSBinstead of primary antibody. An anti-human IgG Fc specific, polyclonal,FITC conjugated antibody was used for all samples as a secondaryantibody.

FIG. 15 depicts the ADCC activity of different C2B8 antibody samples atdifferent antibody concentrations (0.04-5 μg/ml). Sample C2B8-ntrepresents the ADCC activity of the C2B8 antibody produced in a cellline without GnT III expression. Samples C2B8-2000t, C2B8-50t andC2B8-25t show the ADCC activity of three antibody samples produced atdecreasing tetracycline concentrations (i.e., increasing GnT IIIexpression).

VI. DEFINITIONS

Terms are used herein as generally used in the art, unless otherwisedefined in the following:

As used herein, the term antibody is intended to include whole antibodymolecules, antibody fragments, or fusion proteins that include a regionequivalent to the Fc region of an immunoglobulin.

As used herein, the term glycoprotein-modifying glycosyl transferaserefers to an enzyme that effects modification of the glycosylationpattern of a glycoprotein. Examples of glycoprotein-modifying glycosyltransferases include, but are not limited to glycosyl transferases suchas GnT III, GnT V, GalT, and Man II.

As used herein, the term glycosylation engineering is considered toinclude any sort of change to the glycosylation pattern of a naturallyoccurring polypeptide or fragment thereof. Glycosylation engineeringincludes metabolic engineering of the glycosylation machinery of a cell,including genetic manipulations of the oligosaccharide synthesispathways to achieve altered glycosylation of glycoproteins expressed incells. Furthermore, glycosylation engineering includes the effects ofmutations and cell environment on glycosylation.

As used herein, the term host cell covers any kind of cellular systemwhich can be engineered to generate modified glycoforms of proteins,protein fragments, or peptides of interest, including antibodies andantibody fragments. Typically, the host cells have been manipulated toexpress optimized levels of at least one glycoprotein-modifying glycosyltransferase, including, but not limited to GnT III, GnT V, GalT, and ManII, and/or at least one glycosidase. Host cells include cultured cells,e.g., mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells,SP2/0 cells, or hybridoma cells, yeast cells, and insect cells, to nameonly few, but also cells comprised within a transgenic animal orcultured tissue.

As used herein, the term Fc-mediated cellular cytotoxicity is intendedto include antibody dependent cellular cytotoxicity (ADCC), and cellularcytotoxicity directed to those cells that have been engineered toexpress on their cell surface an Fc-region or equivalent region of animmunoglobin G, and cellular cytotoxicity mediated by a soluble fusionprotein consisting of a target protein domain fused to the N-terminus ofan Fc-region or equivalent region of an immunoglobulin G.

VII. DETAILED DESCRIPTION OF THE INVENTION A. General Overview

The objective of the present invention is to provide glycoforms ofproteins, in particular antibodies, including whole antibody molecules,antibody fragments, or fusion proteins that include a region equivalentto the Fc region of an immunoglobulin, to produce new variants of atherapeutic protein. The invention is based, in part, on the inventors'discovery that the glycosylation reaction network of a cell can bemanipulated to maximize the proportion of certain glycoforms within thepopulation, and that certain glycoforms have improved therapeuticcharacteristics. The invention is further based, in part, on thediscovery of ways to identify glycoforms of proteins which have animproved therapeutic value, and how to generate them reproducibly. Theinvention is further based, in part, on the discovery that there is apreferred range of glycoprotein-modifying glycosyl transferaseexpression in the antibody-generating cell, for increasing complexN-linked oligosaccharides carrying bisecting GlcNAc.

As such, the present invention is directed, generally, to methods forthe glycosylation engineering of proteins to alter and improve theirtherapeutic properties. More specifically, the present inventiondescribes methods for producing in a host cell an antibody which has analtered glycosylation pattern resulting in an enhanced antibodydependent cellular cytotoxicity (ADCC). For the practice of the methods,the present invention provides host cells which harbor a nucleic acidencoding an antibody and at least one nucleic acid encoding aglycoprotein-modifying glycosyl transferase. Further, the presentinvention provides methods and protocols of culturing such host cellsunder conditions which permit the expression of the desired antibodyhaving an altered glycosylation pattern resulting in an enhancedantibody dependent cellular cytotoxicity. Further, methods for isolatingthe so generated antibody with enhanced antibody dependent cellularcytotoxicity are described.

In more specific embodiments of the invention, two monoclonalantibodies, namely the anti-neuroblastoma antibody chCE7, and theanti-CD20 antibody C2B8, have been used as model therapeuticglycoproteins, and the target glycoforms have been those carrying aspecial class of carbohydrate, namely bi-antennary complex N-linkedoligosaccharides modified with bisecting N-acetylglucosamine (GlcNAc).In the model system provided by the invention, CHO cells are used ashost cells, although many other cell systems may be contemplated as hostcell system. The glycosyl transferase that adds a bisecting GlcNAc tovarious types of N-linked oligosaccharides, GlcNAc-transferase III (GnTIII), is not normally produced by CHO cells. Stanley and Campell, 1984,J. Biol. Chem. 261:13370-13378.

To investigate the effects of GnT III overexpression experimentally, aCHO cell line with tetracycline-regulated overexpression of a rat GnTIII cDNA was established. Using this experimental system, the inventorsdiscovered that overexpression of GnT III to high levels led to growthinhibition and was toxic to the cells. Another CHO cell line withtetracycline-regulated overexpression of GnT V, which is a distinctglycosyl transferase, showed the same inhibitory effect, indicating thatthis may be a general feature of glycoprotein-modifying glycosyltransferase overexpression. The effect of the enzyme expression on thecell growth sets an upper limit to the level of glycoprotein-modifyingglycosyl transferase overexpression and may therefore also limit theextent to which poorly accessible glycosylation sites can be modified byengineering of glycosylation pathways and patterns using unregulatedexpression vectors.

The production of a set of chCE7 mAb and C2B8 samples differing in theirglycoform distributions by controlling GnT III expression in a rangebetween basal and toxic levels are disclosed. Measurement of the ADCCactivity of the chCE7 mAb samples showed an optimal range of GnT IIIexpression for maximal chCE7 in vitro biological activity. The activitycorrelated with the level of Fc-associated bisected, complexoligosaccharides. Expression of GnT III within the practical range,i.e., where no significant growth inhibition and toxicity are observed,led to an increase of the target bisected, complex structures for thisset of chCE7 samples. The pattern of oligosaccharide peaks inMALDI/TOF-mass spectrometric analysis of chCE7 samples produced at highlevels of GnT III indicates that a significant proportion of potentialGnT III substrates is diverted to bisected hybrid oligosaccharideby-products. Minimization of these by-products by further engineering ofthe pathway could therefore be valuable.

B. Identification and Generation of Nucleic Acids Encoding a Protein forwhich Modification of the Glycosylation Pattern is Desired

The present invention provides host cell systems suitable for thegeneration of altered glycoforms of any protein, protein fragment orpeptide of interest, for which such an alteration in the glycosylationpattern is desired. The nucleic acids encoding such protein, proteinfragment or peptide of interest may be obtained by methods generallyknown in the art. For example, the nucleic acid may be isolated from acDNA library or genomic library. For a review of cloning strategieswhich may be used, see, e.g., Maniatis, 1989, Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press, New York; and Ausubel etal., 1989, Current Protocols in Molecular Biology, (Green PublishingAssociates and Wiley Interscience, New York).

In an alternate embodiment of the invention, the coding sequence of theprotein, protein fragment or peptide of interest may be synthesized inwhole or in part, using chemical methods well known in the art. See, forexample, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233;Crea and Horn, 1980, Nuc. Acids Res. USA 9:2331; Matteucci andCaruthers, 1980, Tetrahedron Letters 21:719; Chow and Kempe, 1981, Nuc.Acids Res. 9:2807-2817. Alternatively, the protein itself could beproduced using chemical methods to synthesize its amino acid sequence inwhole or in part. For example, peptides can be synthesized by solidphase techniques, cleaved from the resin, and purified by preparativehigh performance liquid chromatography. E.g. see Creighton, 1983,Protein Structures And Molecular Principles, W. H. Freeman and Co., NewYork pp. 50-60. The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; see Creighton, 1983, Proteins, Structures andMolecular Principles, W. H. Freeman and Co., New York, pp. 34-49).

In preferred embodiments, the invention provides methods for thegeneration and use of host cell systems for the production of glycoformsof antibodies or antibody fragments or fusion proteins which includeantibody fragments with enhanced antibody-dependent cellularcytotoxicity. Identification of target epitopes and generation ofantibodies having potential therapeutic value, for which modification ofthe glycosylation pattern is desired, and isolation of their respectivecoding nucleic acid sequence is within the scope of the invention.

Various procedures known in the art may be used for the production ofantibodies to target epitopes of interest. Such antibodies include butare not limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments and fragments produced by an Fab expression library. Suchantibodies may be useful, e.g., as diagnostic or therapeutic agents. Astherapeutic agents, neutralizing antibodies, i.e., those which competefor binding with a ligand, substrate or adapter molecule, are ofespecially preferred interest.

For the production of antibodies, various host animals are immunized byinjection with the target protein of interest including, but not limitedto, rabbits, mice, rats, etc. Various adjuvants may be used to increasethe immunological response, depending on the host species, including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to the target of interest may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Kohler and Milstein,1975, Nature 256:495-497, the human B-cell hybridoma technique (Kosboret al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:2026-2030) and the EBV-hybridoma technique (Cole etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producesingle chain antibodies having a desired specificity.

Antibody fragments which contain specific binding sites of the targetprotein of interest may be generated by known techniques. For example,such fragments include, but are not limited to, F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed (Huse et al., 1989, Science 246:1275-1281) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity to the target protein of interest.

Once an antibody or antibody fragment has been identified for whichmodification in the glycosylation pattern are desired, the codingnucleic acid sequence is identified and isolated using techniques wellknown in the art. See, supra.

C. Generation of Cell Lines for the Production of Proteins with AlteredGlycosylation Pattern

The present invention provides host cell expression systems for thegeneration of proteins having modified glycosylation patterns. Inparticular, the present invention provides host cell systems for thegeneration of glycoforms of proteins having an improved therapeuticvalue. Therefore, the invention provides host cell expression systemsselected or engineered to increase the expression of aglycoprotein-modifying glycosyltransferase. Specifically, such host cellexpression systems may be engineered to comprise a recombinant nucleicacid molecule encoding a glycoprotein-modifying glycosyltransferase,operatively linked to a constitutive or regulated promoter system.Alternatively, host cell expression systems may be employed thatnaturally produce, are induced to produce, and/or are selected toproduce a glycoprotein-modifying glycosyltransferase.

In one specific embodiment, the present invention provides a host cellthat has been engineered to express at least one nucleic acid encoding aglycoprotein-modifying glycosyl transferase. In one aspect, the hostcell is transformed or transfected with a nucleic acid moleculecomprising at least one gene encoding a glycoprotein-modifying glycosyltransferase. In an alternate aspect, the host cell has been engineeredand/or selected in such way that an endogenous glycoprotein-modifyingglycosyl transferase is activated. For example, the host cell may beselected to carry a mutation triggering expression of an endogenousglycoprotein-modifying glycosyl transferase. This aspect is exemplifiedin one specific embodiment, where the host cell is a CHO lec10 mutant.Alternatively, the host cell may be engineered such that an endogenousglycoprotein-modifying glycosyl transferase is activated. In againanother alternative, the host cell is engineered such that an endogenousglycoprotein-modifying glycosyl transferase has been activated byinsertion of a regulated promoter element into the host cell chromosome.In a further alternative, the host cell has been engineered such that anendogenous glycoprotein-modifying glycosyl transferase has beenactivated by insertion of a constitutive promoter element, a transposon,or a retroviral element into the host cell chromosome.

Generally, any type of cultured cell line can be used as a background toengineer the host cell lines of the present invention. In a preferredembodiment, CHO cells, BHK cells, NS0 cells, SP2/0 cells, or a hybridomacell line is used as the background cell line to generate the engineeredhost cells of the invention.

The invention is contemplated to encompass engineered host cellsexpressing any type of glycoprotein-modifying glycosyl transferase asdefined herein. However, in preferred embodiments, at least oneglycoprotein-modifying glycosyl transferase expressed by the host cellsof the invention is GnT III, or, alternatively,β(1,4)-N-acetylglucosaminyltransferase V (GnT V). However, also othertypes of glycoprotein-modifying glycosyl transferase may be expressed inthe host system, typically in addition to GnT III or GnT V, includingβ(1,4)-galactosyl transferase (GalT), and mannosidase II (Man II). Inone embodiment of the invention, GnT III is coexpressed with GalT. Inanother embodiment of the invention, GnT III is coexpressed with Man II.In a further embodiment of the invention, GnT III is coexpressed withGalT and Man II. However, any other permutation ofglycoprotein-modifying glycosyl transferases is within the scope of theinvention. Further, expression of a glycosidase in the host cell systemmay be desired.

One or several nucleic acids encoding a glycoprotein-modifying glycosyltransferase may be expressed under the control of a constitutivepromoter or, alternately, a regulated expression system. Suitableregulated expression systems include, but are not limited to, atetracycline-regulated expression system, an ecdysone-inducibleexpression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different nucleic acids encodingglycoprotein-modifying glycosyl transferases are comprised within thehost cell system, some of them may be expressed under the control of aconstitutive promoter, while others are expressed under the control of aregulated promoter. The optimal expression levels will be different foreach protein of interest, and will be determined using routineexperimentation. Expression levels are determined by methods generallyknown in the art, including Western blot analysis using a glycosyltransferase specific antibody, Northern blot analysis using a glycosyltransferase specific nucleic acid probe, or measurement of enzymaticactivity. Alternatively, a lectin may be employed which binds tobiosynthetic products of the glycosyl transferase, for example, E₄-PHAlectin. In a further alternative, the nucleic acid may be operativelylinked to a reporter gene; the expression levels of theglycoprotein-modifying glycosyl transferase are determined by measuringa signal correlated with the expression level of the reporter gene. Thereporter gene may transcribed together with the nucleic acid(s) encodingsaid glycoprotein-modifying glycosyl transferase as a single mRNAmolecule; their respective coding sequences may be linked either by aninternal ribosome entry site (IRES) or by a cap-independent translationenhancer (CITE). The reporter gene may be translated together with atleast one nucleic acid encoding said glycoprotein-modifying glycosyltransferase such that a single polypeptide chain is formed. The nucleicacid encoding the glycoprotein-modifying glycosyl transferase may beoperatively linked to the reporter gene under the control of a singlepromoter, such that the nucleic acid encoding the glycoprotein-modifyingglycosyl transferase and the reporter gene are transcribed into an RNAmolecule which is alternatively spliced into two separate messenger RNA(mRNA) molecules; one of the resulting mRNAs is translated into saidreporter protein, and the other is translated into saidglycoprotein-modifying glycosyl transferase.

If several different nucleic acids encoding a glycoprotein-modifyingglycosyl transferase are expressed, they may be arranged in such waythat they are transcribed as one or as several mRNA molecules. If theyare transcribed as a single mRNA molecule, their respective codingsequences may be linked either by an internal ribosome entry site (IRES)or by a cap-independent translation enhancer (CITE). They may betranscribed from a single promoter into an RNA molecule which isalternatively spliced into several separate messenger RNA (mRNA)molecules, which then are each translated into their respective encodedglycoprotein-modifying glycosyl transferase.

In other embodiments, the present invention provides host cellexpression systems for the generation of therapeutic proteins, forexample antibodies, having an enhanced antibody-dependent cellularcytotoxicity, and cells which display the IgG Fc region on the surfaceto promote Fc-mediated cytotoxicity. Generally, the host cell expressionsystems have been engineered and/or selected to express nucleic acidsencoding the protein for which the production of altered glycoforms isdesired, along with at least one nucleic acid encoding aglycoprotein-modifying glycosyl transferase. In one embodiment, the hostcell system is transfected with at least one gene encoding aglycoprotein-modifying glycosyl transferase. Typically, the transfectedcells are selected to identify and isolate clones that stably expressthe glycoprotein-modifying glycosyl transferase. In another embodiment,the host cell has been selected for expression of endogenous glycosyltransferase. For example, cells may be selected carrying mutations whichtrigger expression of otherwise silent glycoprotein-modifying glycosyltransferases. For example, CHO cells are known to carry a silent GnT IIIgene that is active in certain mutants, e.g., in the mutant Lec10.Furthermore, methods known in the art may be used to activate silentglycoprotein-modifying glycosyl transferase genes, including theinsertion of a regulated or constitutive promoter, the use oftransposons, retroviral elements, etc. Also the use of gene knockouttechnologies or the use of ribozyme methods may be used to tailor thehost cell's glycosyl transferase and/or glycosidase expression levels,and is therefore within the scope of the invention.

Any type of cultured cell line can be used as background to engineer thehost cell lines of the present invention. In a preferred embodiment, CHOcells, BHK cells, NS0 cells, SP2/0 cells. Typically, such cell lines areengineered to further comprise at least one transfected nucleic acidencoding a whole antibody molecule, an antibody fragment, or a fusionprotein that includes a region equivalent to the Fc region of animmunoglobulin. In an alternative embodiment, a hybridoma cell lineexpressing a particular antibody of interest is used as background cellline to generate the engineered host cells of the invention.

Typically, at least one nucleic acid in the host cell system encodes GnTIII, or, alternatively, GnT V. However, also other types ofglycoprotein-modifying glycosyl transferase may be expressed in the hostsystem, typically in addition to GnT III or GnT V, including GalT, andMan II. In one embodiment of the invention, GnT III is coexpressed withGalT. In another embodiment of the invention, GnT III is coexpressedwith Man II. In a further embodiment of the invention, GnT III iscoexpressed with GalT and Man II. However, any other permutation ofglycoprotein-modifying glycosyl transferases is within the scope of theinvention. Further, expression of a glycosidase in the host cell systemmay be desired.

One or several nucleic acids encoding a glycoprotein-modifying glycosyltransferase may be expressed under the control of a constitutivepromoter, or alternately, a regulated expression system. Suitableregulated expression systems include, but are not limited to, atetracycline-regulated expression system, an ecdysone-inducibleexpression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different nucleic acids encodingglycoprotein-modifying glycosyl transferases are comprised within thehost cell system, some of them may be expressed under the control of aconstitutive promoter, while others are expressed under the control of aregulated promoter. The optimal expression levels will be different foreach protein of interest, and will be determined using routineexperimentation. Expression levels are determined by methods generallyknown in the art, including Western blot analysis using a glycosyltransferase specific antibody, Northern blot analysis using a glycosyltransferase specific nucleic acid probe, or measurement of enzymaticactivity. Alternatively, a lectin may be employed which binds tobiosynthetic products of glycosyl transferase, for example, E₄-PHAlectin. In a further alternative, the nucleic acid may be operativelylinked to a reporter gene; the expression levels of theglycoprotein-modifying glycosyl transferase are determined by measuringa signal correlated with the expression level of the reporter gene. Thereporter gene may transcribed together with the nucleic acid(s) encodingsaid glycoprotein-modifying glycosyl transferase as a single mRNAmolecule; their respective coding sequences may be linked either by aninternal ribosome entry site (IRES) or by a cap-independent translationenhancer (CITE). The reporter gene may be translated together with atleast one nucleic acid encoding said glycoprotein-modifying glycosyltransferase such that a single polypeptide chain is formed. The nucleicacid encoding the glycoprotein-modifying glycosyl transferase may beoperatively linked to the reporter gene under the control of a singlepromoter, such that the nucleic acid encoding the glycoprotein-modifyingglycosyl transferase and the reporter gene are transcribed into an RNAmolecule which is alternatively spliced into two separate messenger RNA(mRNA) molecules; one of the resulting mRNAs is translated into saidreporter protein, and the other is translated into saidglycoprotein-modifying glycosyl transferase.

If several different nucleic acids encoding a glycoprotein-modifyingglycosyl transferase are expressed, they may be arranged in such waythat they are transcribed as one or as several mRNA molecules. If theyare transcribed as single mRNA molecule, their respective codingsequences may be linked either by an internal ribosome entry site (IRES)or by a cap-independent translation enhancer (CITE). They may betranscribed from a single promoter into an RNA molecule which isalternatively spliced into several separate messenger RNA (mRNA)molecules, which then are each translated into their respective encodedglycoprotein-modifying glycosyl transferase.

1. Expression Systems

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of theprotein of interest and the coding sequence of theglycoprotein-modifying glycosyl transferase and appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., 1989, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, New York and Ausubel et al.,1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, New York.

A variety of host-expression vector systems may be utilized to expressthe coding sequence of the protein of interest and the coding sequenceof the glycoprotein-modifying glycosyl transferase. Preferably,mammalian cells are used as host cell systems transfected withrecombinant plasmid DNA or cosmid DNA expression vectors containing thecoding sequence of the protein of interest and the coding sequence ofthe glycoprotein-modifying glycosyl transferase. Most preferably, CHOcells, BHK cells, NS0 cells, or SP2/0 cells, or alternatively, hybridomacells are used as host cell systems. In alternate embodiments, othereukaryotic host cell systems may be contemplated, including, yeast cellstransformed with recombinant yeast expression vectors containing thecoding sequence of the protein of interest and the coding sequence ofthe glycoprotein-modifying glycosyl transferase; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the coding sequence of the protein of interest and the codingsequence of the glycoprotein-modifying glycosyl transferase; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence of the protein of interest andthe coding sequence of the glycoprotein-modifying glycosyl transferase;or animal cell systems infected with recombinant virus expressionvectors (e.g., adenovirus, vaccinia virus) including cell linesengineered to contain multiple copies of the DNA encoding the protein ofinterest and the coding sequence of the glycoprotein-modifying glycosyltransferase either stably amplified (CHO/dhfr) or unstably amplified indouble-minute chromosomes (e.g., murine cell lines).

For the methods of this invention, stable expression is generallypreferred to transient expression because it typically achieves morereproducible results and also is more amenable to large scaleproduction. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with therespective coding nucleic acids controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows selection of cells whichhave stably integrated the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al, 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al, 1984, Gene 30:147) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); the glutaminesynthase system; and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, in: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

2. Identification of Transfectants or Transformants that Express theProtein Having a Modified Glycosylation Pattern

The host cells which contain the coding sequence and which express thebiologically active gene products may be identified by at least fourgeneral approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) thepresence or absence of “marker” gene functions; (c) assessing the levelof transcription as measured by the expression of the respective mRNAtranscripts in the host cell; and (d) detection of the gene product asmeasured by immunoassay or by its biological activity.

In the first approach, the presence of the coding sequence of theprotein of interest and the coding sequence of theglycoprotein-modifying glycosyl transferase(s) inserted in theexpression vector can be detected by DNA-DNA or DNA-RNA hybridizationusing probes comprising nucleotide sequences that are homologous to therespective coding sequences, respectively, or portions or derivativesthereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the coding sequence of the protein of interest and the codingsequence of the glycoprotein-modifying glycosyl transferase are insertedwithin a marker gene sequence of the vector, recombinants containing therespective coding sequences can be identified by the absence of themarker gene function. Alternatively, a marker gene can be placed intandem with the coding sequences under the control of the same ordifferent promoter used to control the expression of the codingsequences. Expression of the marker in response to induction orselection indicates expression of the coding sequence of the protein ofinterest and the coding sequence of the glycoprotein-modifying glycosyltransferase.

In the third approach, transcriptional activity for the coding region ofthe protein of interest and the coding sequence of theglycoprotein-modifying glycosyl transferase can be assessed byhybridization assays. For example, RNA can be isolated and analyzed byNorthern blot using a probe homologous to the coding sequences of theprotein of interest and the coding sequence of theglycoprotein-modifying glycosyl transferase or particular portionsthereof. Alternatively, total nucleic acids of the host cell may beextracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the protein products of theprotein of interest and the coding sequence of theglycoprotein-modifying glycosyl transferase can be assessedimmunologically, for example by Western blots, immunoassays such asradioimmuno-precipitation, enzyme-linked immunoassays and the like. Theultimate test of the success of the expression system, however, involvesthe detection of the biologically active gene products.

D. Generation and Use of Proteins and Protein Fragments Having AlteredGlycosylation Patterns 1. Generation and Use of Antibodies HavingEnhanced Antibody-Dependent Cellular Cytotoxicity

In preferred embodiments, the present invention provides glycoforms ofantibodies and antibody fragments having an enhanced antibody-dependentcellular cytotoxicity.

Clinical trials of unconjugated monoclonal antibodies (mAbs) for thetreatment of some types of cancer have recently yielded encouragingresults. Dillman, 1997, Cancer Biother. & Radiopharm. 12:223-225; Deo etal., 1997, Immunology Today 18:127. A chimeric, unconjugated IgGl hasbeen approved for low-grade or follicular B-cell non-Hodgkin's lymphoma(Dillman, 1997, supra), while another unconjugated mAb, a humanized IgGltargeting solid breast tumors, has also been showing promising resultsin phase III clinical trials. Deo et al., 1997, supra. The antigens ofthese two mAbs are highly expressed in their respective tumor cells andthe antibodies mediate potent tumor destruction by effector cells invitro and in vivo. In contrast, many other unconjugated mAbs with finetumor specificities cannot trigger effector functions of sufficientpotency to be clinically useful. Frost et al., 1997, Cancer 80:317-333;Surfus et al., 1996, J. Immunother. 19:184-191. For some of these weakermAbs, adjunct cytokine therapy is currently being tested. Addition ofcytokines can stimulate antibody-dependent cellular cytotoxicity (ADCC)by increasing the activity and number of circulating lymphocytes. Frostet al., 1997, supra; Surfus et al., 1996, supra. ADCC, a lytic attack onantibody-targeted cells, is triggered upon binding of lymphocytereceptors to the constant region (Fc) of antibodies. Deo et al., 1997,supra.

A different, but complementary, approach to increase ADCC activity ofunconjugated IgGls would be to engineer the Fc region of the antibody toincrease its affinity for the lymphocyte receptors (FcγRs). Proteinengineering studies have shown that FcγRs interact with the lower hingeregion of the IgG CH2 domain. Lund et al., 1996, J. Immunol.157:4963-4969. However, FcγR binding also requires the presence ofoligosaccharides covalently attached at the conserved Asn 297 in the CH2region. Lund et al., 1996, supra; Wright and Morrison, 1997, Tibtech15:26-31, suggesting that either oligosaccharide and polypeptide bothdirectly contribute to the interaction site or that the oligosaccharideis required to maintain an active CH2 polypeptide conformation.Modification of the oligosaccharide structure can therefore be exploredas a means to increase the affinity of the interaction.

An IgG molecule carries two N-linked oligosaccharides in its Fc region,one on each heavy chain. As any glycoprotein, an antibody is produced asa population of glycoforms which share the same polypeptide backbone buthave different oligosaccharides attached to the glycosylation sites. Theoligosaccharides normally found in the Fc region of serum IgG are ofcomplex bi-antennary type (Wormald et al., 1997, Biochemistry36:130-1380), with low level of terminal sialic acid and bisectingN-acetylglucosamine (GlcNAc), and a variable degree of terminalgalactosylation and core fucosylation (FIG. 1). Some studies suggestthat the minimal carbohydrate structure required for FcγR binding lieswithin the oligosaccharide core. Lund et al., 1996, supra. The removalof terminal galactoses results in approximately a two-fold reduction inADCC activity, indicating a role for these residues in FcγR receptorbinding. Lund et al., 1996, supra.

The mouse- or hamster-derived cell lines used in industry and academiafor production of unconjugated therapeutic mAbs normally attach therequired oligosaccharide determinants to Fc sites. IgGs expressed inthese cell lines lack, however, the bisecting GlcNAc found in lowamounts in serum IgGs. Lifely et al., 1995, Glycobiology 318:813-822. Incontrast, it was recently observed that a rat myeloma-produced,humanized IgGl (CAMPATH-1H) carried a bisecting GlcNAc in some of itsglycoforms. Lifely et al., 1995, supra. The rat cell-derived antibodyreached a similar in vitro ADCC activity as CAMPATH-1H antibodiesproduced in standard cell lines, but at significantly lower antibodyconcentrations.

The CAMPATH antigen is normally present at high levels on lymphomacells, and this chimeric mAb has high ADCC activity in the absence of abisecting GlcNAc. Lifely et al., 1995, supra. Even though in the studyof Lifely et al., 1995, supra. the maximal in vitro ADCC activity wasnot increased by altering the glycosylation pattern, the fact that thislevel of activity was obtained at relatively low antibody concentrationsfor the antibody carrying bisected oligosaccharides suggests animportant role for bisected oligosaccharides. An approach was developedto increase the ADCC activity of IgGls with low basal activity levels byproducing glycoforms of these antibodies carrying bisectedoligosaccharides in the Fc region.

In the N-linked glycosylation pathway, a bisecting GlcNAc is added bythe enzyme β(1,4)-N-acetylglucosaminyltransferase III (GnT III).Schachter, 1986, Biochem. Cell Biol. 64:163-181. Lifely et al., 1995,supra, obtained different glycosylation patterns of the same antibody byproducing the antibody in different cell lines with different butnon-engineered glycosylation machineries, including a rat myeloma cellline that expressed GnT III at an endogenous, constant level. Incontrast, we used a single antibody-producing CHO cell line, that waspreviously engineered to express, in an externally-regulated fashion,different levels of a cloned GnT III gene. This approach allowed us toestablish for the first time a rigorous correlation between expressionof GnT III and the ADCC activity of the modified antibody.

As demonstrated herein, see, Example 4, infra, C2B8 antibody modifiedaccording to the disclosed method had an about sixteen-fold higher ADCCactivity than the standard, unmodified C2B8 antibody produced underidentical cell culture and purification conditions. Briefly, a C2B8antibody sample expressed in CHO-tTA-C2B8 cells that do not have GnT IIIexpression showed a cytotoxic activity of about 31% (at 1 μg/ml antibodyconcentration), measured as in vitro lysis of SB cells (CD20+) by humanlymphocytes. In contrast, C2B8 antibody derived from a CHO cell cultureexpressing GnT III at a basal, largely repressed level showed at 1 μg/mlantibody concentration a 33% increase in ADCC activity against thecontrol at the same antibody concentration. Moreover, increasing theexpression of GnT III produced a large increase of almost 80% in themaximal ADCC activity (at 1 μg/ml antibody concentration) compared tothe control at the same antibody concentration. See, Example 4, infra.

Further antibodies of the invention having an enhancedantibody-dependent cellular cytotoxicity include, but are not limitedto, anti-human neuroblastoma monoclonal antibody (chCE7) produced by themethods of the invention, a chimeric anti-human renal cell carcinomamonoclonal antibody (ch-G250) produced by the methods of the invention,a humanized anti-HER2 monoclonal antibody produced by the methods of theinvention, a chimeric anti-human colon, lung, and breast carcinomamonoclonal antibody (ING-1) produced by the methods of the invention, ahumanized anti-human 17-1A antigen monoclonal antibody (3622W94)produced by the methods of the invention, a humanized anti-humancolorectal tumor antibody (A33) produced by the methods of theinvention, an anti-human melanoma antibody (R24) directed against GD3ganglioside produced by the methods of the invention, and a chimericanti-human squamous-cell carcinoma monoclonal antibody (SF-25) producedby the methods of the invention. In addition, the invention is directedto antibody fragment and fusion proteins comprising a region that isequivalent to the Fc region of immunoglobulins. See, infra.

2. Generation and use of Fusion Proteins Comprising a Region Equivalentto an Fc Region of an Immunoglobulin that Promote Fc-MediatedCytotoxicity

As discussed above, the present invention relates to a method forenhancing the ADCC activity of therapeutic antibodies. This is achievedby engineering the glycosylation pattern of the Fc region of suchantibodies, in particular by maximizing the proportion of antibodymolecules carrying bisected complex oligosaccharides N-linked to theconserved glycosylation sites in their Fc regions. This strategy can beapplied to enhance Fc-mediated cellular cytotoxicity against undesirablecells mediated by any molecule carrying a region that is an equivalentto the Fc region of an immunoglobulin, not only by therapeuticantibodies, since the changes introduced by the engineering ofglycosylation affect only the Fc region and therefore its interactionswith the Fc receptors on the surface of effector cells involved in theADCC mechanism. Fc-containing molecules to which the presently disclosedmethods can be applied include, but are not limited to, (a) solublefusion proteins made of a targeting protein domain fused to theN-terminus of an Fc-region (Chamov and Ashkenazi, 1996, TIBTECH 14: 52)and (b) plasma membrane-anchored fusion proteins made of a type IItransmembrane domain that localizes to the plasma membrane fused to theN-terminus of an Fc region (Stabila, P. F., 1998, Nature Biotech. 16:1357).

In the case of soluble fusion proteins (a) the targeting domain directsbinding of the fusion protein to undesirable cells such as cancer cells,i.e., in an analogous fashion to therapeutic antibodies. The applicationof presently disclosed method to enhance the Fc-mediated cellularcytotoxic activity mediated by these molecules would therefore beidentical to the method applied to therapeutic antibodies. See, Example2 of United States Provisional Application Ser. No. 60/082,581,incorporated herein by reference.

In the case of membrane-anchored fusion proteins (b) the undesirablecells in the body have to express the gene encoding the fusion protein.This can be achieved either by gene therapy approaches, i.e., bytransfecting the cells in vivo with a plasmid or viral vector thatdirects expression of the fusion protein-encoding gene to undesirablecells, or by implantation in the body of cells genetically engineered toexpress the fusion protein on their surface. The later cells wouldnormally be implanted in the body inside a polymer capsule (encapsulatedcell therapy) where they cannot be destroyed by an Fc-mediated cellularcytotoxicity mechanism. However should the capsule device fail and theescaping cells become undesirable, then they can be eliminated byFc-mediated cellular cytotoxicity. Stabila et al, 1998, Nature Biotech.16: 1357. In this case, the presently disclosed method would be appliedeither by incorporating into the gene therapy vector an additional geneexpression cassette directing adequate or optimal expression levels ofGnT III or by engineering the cells to be implanted to express adequateor optimal levels of GnT III. In both cases, the aim of the disclosedmethod is to increase or maximize the proportion of surface-displayed Fcregions carrying bisected complex oligosaccharides.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

VIII. EXAMPLES A. Example 1 Tetracycline-Regulated Overexpression ofGlycosyl Transferases in Chinese Hamster Ovary Cells

To establish a cell line in which the expression of GnT III could beexternally-controlled, a tetracycline-regulated expression system wasused. Gossen, M. and Bujard, H., 1992, Proc. Nat. Acad. Sci. USA, 89:5547-5551. The amount of GnT III in these cells could be controlledsimply by manipulating the concentration of tetracycline in the culturemedium. Using this system, it was found that overexpression of GnT IIIto high levels led to growth inhibition and was toxic to the cells.Another CHO cell line with tetracycline-regulated overexpression of GnTV, a distinct glycoprotein-modifying glycosyl transferase, showed thesame inhibitory effect, indicating that this may be a general feature ofglycoprotein-modifying glycosyl transferase overexpression. Thisphenomenon has not been reported previously, probably due to the factthat investigators generally have used constitutive promoters forrelated experiments. The growth effect sets an upper limit to the levelof glycoprotein-modifying glycosyl transferase overexpression, and maythereby also limit the maximum extent of modification of poorlyaccessible glycosylation sites.

1. Materials and Methods

Establishment Of CHO Cells With Tetracycline-Regulated Expression OfGlycosyltransferases. In a first step, an intermediate CHO cell line(CHO-tTA) was first generated that constitutively expresses atetracycline-controlled transactivator (tTA) at a level for the adequatefor the regulation system. Using Lipofectamine reagent (Gibco,Eggenfelden, Germany), CHO (DUKX) cells were co-transfected, withpUHD15-1, a vector for constitutive expression of the tTA gene (Gossenand Bujard, 1992, Proc. Nat. Acad. Sci. USA, 89: 5547-5551), andpSV2Neo, a vector for constitutive expression of a neomycin resistancegene (Clontech, Palo Alto, Calif.). Stable, drug-resistant clones wereselected and screened for adequate levels of tTA expression viatransient transfections with a tetracycline-regulated β-galactosidaseexpression vector, pUHG16-3. C-myc epitope-encoding DNA was added to the3′ end of the rat GnT III cDNA (Nishikawa et al., 1992, J. Biol. Chem.267:18199-18204) by PCR amplification. Nilsson et al, 1993, J. CellBiol. 120:5-13. The product was sequenced and subcloned into pUHD10-3, avector for tetracycline-regulated expression (Gossen and Bujard, supra)to generate the vector pUHD10-3-GnT IIIm. The human GnT V cDNA (Saito etal., 1995, Eur. J. Biochem. 233:18-26), was directly subcloned intopUHD10-3 to generate plasmid vector pUHD10-3-GnT V. CHO-tTA cells wereco-transfected using a calcium phosphate transfection method (Jordan andWurm, 1996, Nucleic Acids Res. 24:596-601), with pPur, a vector forconstitutive expression of puromycin resistance (Clontech, Palo Alto,Calif.), and either the vector pUHD10-3-GnT IIIm or the vectorpUHD10-3-GnT V. Puromycin resistant clones were selected in the presenceof tetracycline, isolated and then analyzed for tetracycline-regulatedexpression of GnT III or GnT V via western blots analysis. See, infra.

Western And Lectin Blotting. For Western blot analysis of GnT III or GnTV, cell lysates were separated by SDS-PAGE and electroblotted to PVDFmembranes (Millipore, Bedford, Mass.). GnT III was detected using theanti-c-myc monoclonal antibody 9E10 (Nilsson et al, 1993, J. Cell Biol.120:5-13) and GnT V using with an anti-GnT V rabbit polyclonal antibody(Chen et al., 1995, Glycoconjugate J. 12:813-823). Anti-mouse oranti-rabbit IgG-horse radish peroxidase (Amersham, Arlington, Ill.) wasused as secondary antibody. Bound secondary antibody was detected usingan enhanced chemiluminescence kit (ECL kit, Amersham, Arlington, Ill.)

For lectin blot analysis of glycoproteins modified either by GnT III- orGnT V-catalyzed reactions, biotinylated E-PHA (Oxford Glycosciences,Oxford, United Kingdom) or L-PHA-digoxigenin (Boehringer Mannheim,Mannheim, Germany), respectively, were used. Merkle and Cummings, 1987,Methods Enzymol. 138:232-259.

2. Results and Discussion

Establishment Of CHO Cell Lines With Tetracycline-RegulatedOverexpression Of Glycosyl Transferases. The strategy used forestablishment of glycosyl transferase overexpressing cell linesconsisted of first generating an intermediate CHO cell lineconstitutively expressing the tetracycline-controlled transactivator(tTA) at an adequate level for the system to work. Yin et al., 1996,Anal. Biochem. 235:195-201. This level had to be high enough to activatehigh levels of transcription, in the absence of tetracycline, from theminimal promoter upstream of the glycosyl transferase genes. CHO cellswere co-transfected with a vector for constitutive expression for tTA,driven by the human cytomegalovirus. (hCMV) promoter/enhancer, and avector for expression of a neomycin-resistance (Neo^(R)) gene. An excessof the tTA-expression vector was used and neomycin-resistant clones wereisolated.

In mammalian cells, co-transfected DNA integrates adjacently at randomlocations within the chromosomes, and expression depends to a largeextent on the site of integration and also on the number of copies ofintact expression cassettes. A mixed population of clones with differentexpression levels of the transfected genes is generated. Yin et al.,1996, supra. Selection for neomycin resistance merely selects forintegration of an intact Neo^(R) expression cassette, while the use ofan excess of the tTA-expression vector increases the probability offinding clones with good expression of tTA. The mixed population ofclones has to be screened using a functional assay for tTA expression.Gossen and Bujard, 1992, supra; Yin et al., 1996, supra. This was doneby transfection of each clone with a second vector harboring a reportergene, lacZ, under the control of the tet-promoter and screening fortetracycline-regulated (tet-regulated), transient expression (i.e., oneto three days after transfection) of β-galactosidase activity. CHOt17,which showed the highest level of tet-regulated β-galactosidase activityamong twenty screened clones, was selected for further work.

CHOt17 cells were tested for tet-regulated expression of GnT III bytransfecting the cells with vector pUHD1O-3-GnT IIIm and comparing therelative levels of GnT III after incubation of the cells in the presenceand absence of tetracycline for 36 h. GnT III levels were compared bywestern blot analysis, using a monoclonal antibody (9E10) whichrecognizes the c-myc peptide epitope tag at the carboxy-terminus of GnTIII. The tag had been introduced through a modification of the glycosyltransferase gene using PCR amplification. Various reports havedemonstrated addition of peptide epitope tags to the carboxy-termini ofglycosyl transferases, a group of enzymes sharing the same topology,without disruption of localization or activity. Nilsson et al., 1993,supra; Rabouille et al., 1995, J. Cell Science 108:1617-1627. FIG. 2shows that in clone CHOt17 GnT III accumulation is significantly higherin the absence than in the presence of tetracycline. An additionalclone, CHOt2, which gave weaker activation of transcription in theb-galactosidase activity assay, was tested in parallel (FIG. 2). GnT IIIand β-galactosidase expression levels follow the same pattern oftetracycline-regulation for both of these clones. The range oftetracycline concentrations where GnT III expression can bequantitatively controlled was found to be from 0 to 100 ng/ml (FIG. 3).This result agrees with previous research using different cell lines andgenes (Yin et al., 1996, supra).

To generate a stable cell line with tet-regulated expression of GnT III,CHOt17 cells were co-transfected with vector pUHD1O-3-GnT IlIm andvector, pPUR, for expression of a puromycin resistance gene. Inparallel, CHOt17 cells were co-transfected with pUHD1O-3-GnT V and pPURvectors to generate an analogous cell line for this other glycosyltransferase. A highly efficient calcium phosphate transfection methodwas used and the DNA was linearized at unique restriction sites outsidethe eucaryotic expression cassettes, to decrease the probability ofdisrupting these upon integration. By using a host in which the levelsof tTA expressed had first been proven to be adequate, the probabilityof finding clones with high expression of the glycosyl transferases inthe absence of tetracycline is increased.

Stable integrants were selected by puromycin resistance, keepingtetracycline in the medium throughout clone selection to maintainglycosyl transferase expression at basal levels. For each glycosyltransferase, sixteen puromycin resistant clones were grown in thepresence and absence of tetracycline, and eight of each were analyzed bywestern blot analysis (FIG. 4). The majority of the clones showed goodregulation of glycosyl transferase expression. One of the GnTIII-expressing clones showed a relatively high basal level in thepresence of tetracycline (FIG. 4B, clone 3), which suggests integrationof the expression cassete close to an endogenous CHO-cell enhancer;while two puromycin-resistant clones showed no expression of GnT III inthe absence of tetracycline (FIG. 4B, clones 6 and 8). Among the clonesshowing good regulation of expression, different maximal levels ofglycosyl transferase were observed. This may be due to variations in thesite of integration or number of copies integrated. Activity of theglycosyl transferases was verified by E-PHA and L-PHA lectin binding toendogenous cellular glycoproteins derived from various clones grown inthe presence and absence of tetracycline (FIG. 5). Lectins are proteinswhich bind to specific oligosaccharide structures. E-PHA lectin binds tobisected oligosaccharides, the products of GnT III-catalyzed reactions,and L-PHA binds to tri- and tetra-antennary oligosaccharides produced byGnT V-catalyzed reactions (Merkle and Cummings, 1987, Methods Enzymol.138:232-259). For each glycosyl transferase, a clone with highexpression in the absence, but with undetectable expression in thepresence, of tetracycline (clone 6, FIG. 4A, CHO-tet-GnT V, and clone 4,FIG. 4B, CHO-tet-GnT IlIm) was selected for further work.

B. Example 2 Inhibition of Cell Growth Effected by Glycosyl TransferaseOverexpresseion

During screening of GnT III- and GnT V-expressing clones in the absenceof tetracycline, see, Example 1, supra, approximately half of each setof clones showed a strong inhibition of growth. The extent ofgrowth-inhibition varied among clones, and comparison with expressionlevels estimated from western blot analysis (FIG. 4) suggested acorrelation between the degree of growth-inhibition and glycosyltransferase overexpression. This correlation was firmly established bygrowing the final clones, CHO-tet-GnT IlIm and CHO-tet-GnT V, indifferent concentrations of tetracycline. A strong inhibition of growthwas evident after two days of culture at low levels of tetracycline(FIG. 6). Growth-inhibited cells displayed a small, rounded morphologyinstead of the typical extended shape of adherent CHO cells. After a fewdays, significant cell death was apparent from the morphology of thegrowth-inhibited cells.

Growth-inhibition due to glycosyl transferase overexpression has nothitherto been reported in the literature, probably due to the widespreaduse of constitutive promoters. Those clones giving constitutiveexpression of a glycosyl transferase at growth-inhibiting levels, wouldbe lost during the selection procedure. This was avoided here by keepingtetracycline in the medium, i.e., basal expression levels, throughoutselection. Prior to selection, the frequency of clones capable ofexpressing glycosyl transferases to growth-inhibiting levels usingtraditional mammalian vectors based on the constitutive hCMVpromoter/enhancer would be expected to be lower. This is due to the factthat, for any given gene, the pUHD1O-3 vector in CHO cell lines selectedfor high constitutive levels of tTA, gives significantly higherexpression levels than constitutive hCMV promoter/enhancer-basedvectors, as observed by others. Yin et al., 1996, supra.

Inhibition of cell growth could be due to a direct effect ofoverexpression of membrane-anchored, Golgi-resident glycosyltransferases independent of their in vivo catalytic activity, e.g., viamisfolding in the endoplasmic reticulum (ER) causing saturation ofelements which assist protein folding in the ER. This could possiblyaffect the folding and secretion of other essential cellular proteins.Alternatively, inhibition of growth could be related to increased invivo activity of the glycosyl transferase leading to a change of theglycosylation pattern, in a function-disrupting fashion, of a set ofendogenous glycoproteins necessary for growth under standard in vitroculture conditions.

Independent of the underlying mechanism, the growth-inhibition effecthas two consequences for engineering the glycosylation of animal cells.First, it implies that cotransfection of constitutive glycosyltransferase expression vectors together with vectors for the targetglycoprotein product is a poor strategy. Other ways of linkingexpression of these two classes of proteins, e.g., through the use ofmultiple constitutive promoters of similar strength or use ofmulticistronic, constitutive expression vectors, should also be avoided.In these cases, clones with very high, constitutive expression of thetarget glycoprotein, a pre-requisite for an economical bioprocess, wouldalso have high expression of the glycosyl transferase and would beeliminated during the selection process. Linked, inducible expressioncould also be problematic for industrial bioprocesses, since theviability of the growth-arrested cells would be compromised by theoverexpression of the glycosyl transferase.

The second consequence is that it imposes an upper limit on glycosyltransferase overexpression for glycosylation engineering approaches.Clearly, the conversions of many glycosyl transferase-catalyzedreactions in the cell, at the endogenous levels of glycosyltransferases, are very high for several glycosylation sites. However,glycosylation sites where the oligosaccharides are somewhat inaccessibleor are stabilized in unfavorable conformations for specific glycosyltranferases also exist. For example, it has been observed that additionof bisecting GlcNAc is more restricted to the oligosaccharides attachedto the Fc region than to those located on the variable regions of humanIgG antibodies. Savvidou et al., 1984, Biochemistry 23:3736-3740.Glycosylation engineering of these restricted sites could be affected bysuch a limit on glycosyl transferase expression. Although this wouldimply aiming for an “unnatural” distribution of glycoforms, these couldbe of benefit for special therapeutic applications of glycoproteins.

C. Example 3 Engineering the Glycosylation of an Anti-HumanNeuroblastoma Antibody in Chinese Hamster Ovary Cells

In order to validate the concept of engineering a therapeutic antibodyby modifying its glycosylation pattern, a chimeric anti-humanneuroblastoma IgGl (chCE7) was chosen which has insignificant ADCCactivity when produced by SP2/0 recombinant mouse myeloma cells. ChCE7recognizes a tumor-associated 190-kDa membrane glycoprotein and reactsstrongly with all neuroblastoma tumors tested to date. It has a highaffinity for its antigen (K_(d)of 10¹⁰M⁻¹) and, because of its hightumor-specificity, it is routinely used as a diagnostic tool in clinicalpathology. Amstutz et al., 1993, Int. J. Cancer 53:147-152. In recentstudies, radiolabelled chCE7 has shown good tumor localization in humanpatients. Dürr, 1993, Eur. J. Nucl. Med. 20:858. The glycosylationpattern of chCE7, an anti-neuroblastoma therapeutic monoclonal antibody(mAb) was engineered in CHO cells with tetracycline-regulated expressionof GnT III. A set of mAb samples differing in their glycoformdistribution was produced by controlling GnT III expression in a rangebetween basal and toxic levels, and their glycosylation profiles wereanalyzed by MALDI/TOF-MS of neutral oligosaccharides. Measurement of theADCC activity of these samples showed an optimal range of GnT IIIexpression for maximal chCE7 in vitro biological activity, and thisactivity correlated with the level of Fc-associated bisected, complexoligosaccharides.

1. Materials and Methods

Construction Of chCE7 Expression Vectors. Plasmid vectors 10CE7VH and98CE7VL, for expression of heavy (IgGl) and light (kappa) chains,respectively, of anti-human neuroblastoma chimeric antibody chCE7, whichcontain chimeric genomic DNA including the mouse immunoglobulinpromoter/enhancer, mouse antibody variable regions, and human antibodyconstant regions (Amstutz et al., 1993, Int. J. Cancer 53:147-152) wereused as starting materials for the construction of the final expressionvectors, pchCE7H and pchCE7L. Chimeric heavy and light chain chCE7 geneswere reassembled and subcloned into the pcDNA3.1 (+) vector. Duringreassembly, all introns were removed, the leader sequences were replacedwith synthetic ones, Reff et al., 1994, Blood 83:435-445, and uniquerestriction sites joining the variable and constant region sequenceswere introduced. Introns from the heavy constant region were removed bysplicing with overlap-extension-PCR. Clackson et al., 1991, GeneralApplications of PCR to Gene Cloning and Manipulation, p. 187-214, in:McPherson et al. (ed.), PCR a Practical Approach, Oxford UniversityPress, Oxford.

Production Of chCE7 In CHO Cells Expressing Different Levels Of GnT III.CHO-tet-GnT. IIIm (see, supra) cells were co-transfected with vectorspchCE7H, pchCE7L, and pZeoSV2 (for Zeocin resistance, Invitrogen,Groningen, The Netherlands) using a calcium phosphate transfectionmethod. Zeocin resistant clones were transferred to a 96-well cellculture plate and assayed for chimeric antibody expression using anELISA assay specific for human IgG constant region. Lifely et al, 1995,supra. Four chCE7 antibody samples were derived from parallel culturesof a selected clone (CHO-tet-GnT IIIm-chCE7), grown in FMX-8 cellculture medium supplemented with 10% FCS; each culture containing adifferent level of tetracycline and therefore expressing GnT III atdifferent levels. CHO-tet-GnT IIIm-chCE7 cells were expanded andpreadapted to a different concentration of tetracycline during 7 days.The levels of tetracycline were 2000, 60, 30, and 15 ng/ml.

Purification Of chCE7 Antibody Samples. Antibody was purified fromculture medium by Protein A affinity chromatography on a 1 ml HiTrapProtein A column (Pharmacia Biotech, Uppsala, Sweden), using linear pHgradient elution from 20 mM sodium phosphate, 20 mM sodium citrate, 500mM sodium chloride, 0.01% Tween 20, 1M urea, pH 7.5 (buffer A) to bufferB (buffer A without sodium phosphate, pH 2.5). Affinity purified chCE7samples were buffer exchanged to PBS on a 1 ml ResourceS cation exchangecolumn (Pharmacia Biotech, Uppsala, Sweden). Final purity was judged tobe higher than 95% from SDS-PAGE and Coomasie-Blue staining. Theconcentration of each sample was estimated from the absorbance at 280nm.

Binding Of Antibodies To Neuroblastoma Cells. Binding affinity to humanneuroblastoma cells was estimated from displacement of ¹²⁵I-labeledchCE7 by the CHO-produced samples. Amstutz et al, 1993, supra.

Oligosaccharide Analysis By MALDI/TOF-MS. CE7-2000t, -60t, -30t, and-15t samples were treated with A. urefaciens sialidase (OxfordGlycosciences, Oxford, United Kingdom), following the manufacturer'sinstructions, to remove any sialic acid monosaccharide residues. Thesialidase digests were then treated with peptide N-glycosidase F(PNGaseF, Oxford Glycosciences, Oxford, United Kingdom), following themanufacturer's instructions, to release the N-linked oligosaccharides.Protein, detergents, and salts were removed by passing the digeststhrough microcolumns containing, from top to bottom, 20 ml of SepPak C18reverse phase matrix (Waters, Milford, Mass.), 20 ml of Dowex AG 50W×8cation exchange matrix (BioRad, Hercules, Calif.), and 20 ml of AG 4×4anion exchange matrix (BioRad, Hercules, Calif.). The microcolumns weremade by packing the matrices in a Gel Loader tip (Eppendorf, Basel,Switzerland) filled with ethanol, followed by an equilibration withwater. Küster et al., 1997, Anal. Biochem. 250:82-101. Flow throughliquid and a 300 ml-water wash were pooled, filtered, evaporated todryness at room temperature, and resuspended in 2 ml of deionized water.One microliter was applied to a MALDI-MS sample plate (PerseptiveBiosystems, Farmingham, Mass.) and mixed with 1 ml of a 10 mg/mldehydrobenzoic acid (DHB, Aldrich, Milwaukee, Wis.) solution inacetonitrile. The samples were air dried and the resulting crystals weredissolved in 0.2 ml of ethanol and allowed to recrystallize by airdrying. Harvey, 1993, Rapid Mass. Spectrom. 7:614-619. Theoligosaccharide samples were then analyzed by matrix-assisted laserdesorption ionization/time-of-flight-mass spectrometry (MALDI/TOF-MS)using an Elite Voyager 400 spectrometer (Perseptive Biosystems,Farmingham, Mass.), equipped with a delayed ion extraction MALDI-ionsource, in positive ion and reflector modes, with an accelerationvoltage of 20 kV. One hundred and twenty eight scans were averaged.Bisected biantennary complex oligosaccharide structures were assigned tofive-HexNAc-associated peaks. Non-bisected tri-antennary N-linkedoligosaccharides, the alternative five HexNAc-containing isomers, havenever been found in the Fc region of IgGs and their syntheses arecatalyzed by glycosyltransferases discrete from GnT III.

ADCC Activity Assay. Lysis of IMR-32 human neuroblastoma cells (target)by human lymphocytes (effector), at a target:effector ratio of 1:19,during a 16 h incubation at 37° C. in the presence of differentconcentrations of chCE7 samples, was measured via retention of afluorescent dye. Kolber et al, 1988, J. Immunol. Methods 108: 255-264.IMR-32 cells were labeled with the fluorescent dye Calcein AM for 20 min(final concentration 3.3 μM). The labeled cells (80,000 cells/well) wereincubated for 1 h with different concentrations of CE7 antibody. Then,monocyte depleted mononuclear cells were added (1,500,000 cells/well)and the cell mixture was incubated for 16 h at 37° C. in a 5% C0₂atmosphere. The supernatant was discarded and the cells were washed oncewith HBSS and lysed in Triton X-100 (0.1%). Retention of the fluorescentdye in IMR-32 cells was measured with a fluorometer (Perkin Elmer,Luminscence Spectrometer LS 50B, (Foster City, Calif.) and specificlysis was calculated relative to a total lysis control, resulting fromexposure of the target to a detergent instead of exposure to antibody.The signal in the absence of antibody was set to 0% cytotoxicity. Eachantibody concentration was analyzed by triplicate, and the assay wasrepeated three separate times.

2. Results and Discussion

Production Of chCE7 In CHO Cells Expressing Different Levels Of GnT III.ChCE7 heavy and light chain expression vectors were constructedincorporating the human cytomegalovirus (hCMV) promoter, the bovinegrowth hormone termination and polyadenylation sequences, andeliminating all heavy and light chain introns. This vector design wasbased on reports of reproducible high-level expression of recombinantIgG genes in CHO cells. Reff et al., 1994, supra; Trill et al., 1995,Current Opinion Biotechnol. 6:553-560. In addition, a unique restrictionsites was introduced in each chain, at the junction between the variableand constant regions. These sites conserve the reading frame and do notchange the amino acid sequence. They should enable simple exchange ofthe mouse variable regions, for the production of other mouse-humanchimeric antibodies. Reff et al., 1994, supra. DNA sequencing confirmedthat the desired genes were appropriately assembled, and production ofthe chimeric antibody in transfected CHO cells was verified with a humanFc-ELISA assay.

CHO-tet-GnT IIIm-chCE7 cells, with stable, tetracycline-regulatedexpression of GnT III and stable, constitutive expression of chCE7, wereestablished and scaled-up for production of a set of chCE7 samples.During scale-up, four parallel cultures derived from the same CHO clonewere grown, each at a different level of tetracycline and therefore onlydiffering in the level of expression of the GnT III gene. This procedureeliminates any clonal effects from other variables affecting N-linkedglycoform biosynthesis, permitting a rigorous correlation to beestablished between GnT III gene expression and biological activity ofthe glycosylated antibody. The tetracycline concentration ranged from2000 ng/ml, i.e., the basal level of GnT III expression, to 15 ng/ml, atwhich significant growth inhibition and toxicity due to glycosyltransferase overexpression was observed (see, supra). Indeed, only asmall amount of antibody could be recovered from the latter culture. Thesecond highest level of GnT III expression, using tetracycline at aconcentration of 30 ng/ml, produced only a mild inhibition of growth.The purified antibody yield from this culture was approximately 70% thatfrom the remaining two lower levels of GnT III gene overexpression.

The four antibody samples, CE7-2000t, -60t, -30t, and -15t, numbersdenoting the associated concentration of tetracycline, were purified byaffinity chromatography on Protein A and buffer exchanged to PBS using acation exchange column. Purity was higher than 95% as judged fromSDS-PAGE with Coomassie Blue staining. Binding assays to humanneuroblastoma cells revealed high affinity to the cells and nosignificant differences in antigen binding among the different samples(estimated equilibrium dissociation constants varied between 2.0 and2.7×10⁻¹⁰M). This was as expected, since there are no potential N-linkedglycosylation sites in the CE7 variable regions.

Oligosaccharide Distributions And Levels Of Bisected ComplexOligosaccharides Of Different chCE7 Samples. Oligosaccharide profileswere obtained by matrix-assisted laser desorption/ionization massspectrometry on a time-of-flight instrument (MALDI/TOF-MS). Mixtures ofneutral N-linked oligosaccharides derived from each of the fourCHO-produced antibody samples and from a SP2/0 mouse myeloma-derivedchCE7 (CE7-SP2/0) sample were analyzed using 2,5-dehydrobenzoic acid(2,5-DHB) as the matrix (FIG. 9). Under these conditions, neutraloligosaccharides appear essentially as single [M+Na⁺] ions, which aresometimes accompanied by smaller [M+K⁺] ions, depending on the potassiumcontent of the matrix. Bergweffet al., 1995, Glycoconjugate J.12:318-330.

This type of analysis yields both the relative proportions of neutraloligosaccharides of different mass, reflected by relative peak height,and the isobaric monosaccharide composition of each peak. Küster et al.,1997, supra; Naven and Harvey, 1996, Rapid Commun. Mass Spectrom.10:1361-1366. Tentative structures are assigned to peaks based on themonosaccharide composition, knowledge of the biosynthetic pathway, andon previous structural data for oligosaccharides derived from the sameglycoprotein produced by the same host, since the protein backbone andthe cell type can have a strong influence on the oligosaccharidedistribution. Field et al., 1996, Anal. Biochem. 239:92-98. In the caseof Fc-associated oligosaccharides, only bi-antennary complexoligosaccharides have been detected in IgGs present in human serum orproduced by mammalian cell cultures under normal conditions. Wormald etal., 1997, Biochemistry 36:1370-1380; Wright and Morrison, 1997, Tibtech15:26-31. The pathway leading to these compounds is illustrated in FIG.10, including the mass of the [M+Na⁺] ion corresponding to eacholigosaccharide. High mannose oligosaccharides have also been detectedon antibodies produced in the stationary and death phases of batch cellcultures. Yu Ip et al., 1994, Arch. Biochem. Biophys. 308:387-399.

The two major peaks in the CE7-SP2/0 sample (FIG. 9A) correspond tomasses of fucosylated oligosaccharides with four N-acetylhexosamines(HexNAcs) containing either three (m/z 1486) or four (m/z 1648) hexoses.See, FIG. 10, but note that the summarized notation for oligosaccharidesin this figure does not count the two GlcNAcs of the core. Thiscomposition is consistent with core fucosylated, bi-antennary complexoligosaccharide structures carrying zero or one galactose residues,respectively, typical of Fc-associated oligosaccharides, and aspreviously observed in NMR analysis of Fc oligosaccharides derived froma chimeric IgGl expressed in SP2/0 cells. Bergweff et al., 1995, supra.

GnT III-catalyzed transfer of a bisecting GlcNAc to these bi-antennarycompounds, which are the preferred GnT III acceptors, would lead tooligosaccharides with five HexNAcs (m/z 1689 and 1851, non- andmono-galactosylated, respectively, FIG. 10), which are clearly absent inthe CE7-SP2/0 sample. The latter peaks appear when chCE7 is expressed inCHO-tet-GnTIIIm cells. In the CHO-expressed antibodies the fourHexNAc-containing peaks are also mainly fucosylated, although a smallamount of non-fucosylated structures is evident from the peak at m/z1339 (see, FIG. 10). The level of galactosylation is also not verydifferent between the CHO- and SP2/0-derived material. At the basallevel of GnT III expression (CE7-2000t sample, FIG. 9B), the moleculeswith five HexNAcs are present in a lower proportion than those with fourHexNAcs. A higher level of GnT III expression (CE7-60t sample, FIG. 9C)led to a reversal of the proportions in favor of oligosaccharides withfive HexNAcs. Based on this trend, bisected, bi-antennary complexoligosaccharide structures can be assigned to compounds with fiveHexNAcs in these samples. Tri-antennary N-linked oligosaccharides, thealternative five HexNAc-containing isomers, have never been found in theFc region of IgGs and their syntheses are catalyzed byGlcNAc-transferases discrete from GnT III.

A further increase in GnT III expression (CE7-30t sample, FIG. 9D) didnot lead to any significant change in the levels of bisected complexoligosaccharides. Another peak (m/z 1543) containing five HexNAcsappears at low, but relatively constant levels in the CHO-GnTIII samplesand corresponds in mass to a non-fucosylated, bisected-complexoligosaccharide mass (FIG. 10). The smaller peaks at m/z 1705 and 1867,also correspond to five HexNAc-containing bi-antennary complexoligosaccharides. They can be assigned either to potassium adducts ofthe peaks at m/z 1689 and 1851 (mass difference of 16 Da with respect tosodium adducts) (Küster et al., 1997, supra) or to mono- andbi-galactosylated, bisected complex oligosaccharides without fucose(FIG. 10). Together, the bisected complex oligosaccharides amount toapproximately 25% of the total in sample CE7-2000t and reachapproximately 45 to 50% in samples CE7-60t and CE7-30t.

Additional information From The Oligosaccharide Profiles Of chCE7Samples. Although the levels of bisected complex oligosaccharides werenot higher in sample CE730t, increased overexpression of GnT III didcontinue to reduce, albeit to a small extent, the proportions ofsubstrate bi-antennary complex oligosaccharide substrates. This wasaccompanied by moderate increases in two different, fourHexNAc-containing peaks (m/z 1664 and 1810). The latter two peaks cancorrespond either to galactosylated bi-antennary complexoligosaccharides or to bisected hybrid compounds (FIG. 11). Acombination of both classes of structures is also possible. The relativeincrease in these peaks is consistent with the accumulation of bisectedhybrid by-products of GnT III overexpression. Indeed, the sampleproduced at the highest level of GnT III overexpression, CE7-15t, showeda large increase in the peak at m/z 1664, a reduction in the peak at m/z1810 and a concomitant reduction of complex bisected oligosaccharides toa level of approximately 25%. See, peaks with m/z 1689 and 1851 in FIG.9E and the corresponding structures in FIG. 11. Higher accumulation ofnon-fucosylated (m/z 1664) bisected hybrid by-products, instead offucosylated ones (m/z 1810), would agree with the fact thatoligosaccharides which are first modified by GnT III can no longer bebiosynthetic substrates for core α1,6-fucosyltransferase. Schachter,1986, Biochem. Cell Biol. 64:163-181.

The peak at m/z 1257 is present at a level of 10-15% of the total in theCHO-derived samples and at a lower level in CE7-SP2/0 (FIG. 9). Itcorresponds to five hexoses plus two HexNAcs. The only known N-linkedoligosaccharide structure with this composition is a fivemannose-containing compound of the high-mannose type. Another highmannose oligosaccharide, a six mannose one (m/z 1420), is also presentat much lower levels. As mentioned above, such oligosaccharides havebeen detected in the Fc of IgGs expressed in the late phase of batchcell cultures. Yu Ip et al., 1994, supra.

Antibody Dependent Cellular Cytotoxicity Of chCE7 Samples. ChCE7 showssome ADCC activity, measured as in vitro lysis of neuroblastoma cells byhuman lymphocytes, when expressed in CHO-tet-GnTIIIm cells with theminimum level of GnT III overexpression (FIG. 12, sample CE7-2000t).Raising the level of GnT III produced a large increase in ADCC activity(FIG. 12, sample CE7-60t). Further overexpression of GnT III was notaccompanied by an additional increase in activity (FIG. 12, sampleCE7-30t), and the highest level of expression actually led to reducedADCC (FIG. 12, sample CE7-15t). Besides exhibiting the highest ADCCactivities, both CE7-60t and CE7-30t samples show significant levels ofcytotoxicity at very low antibody concentrations. These results showthat there is an optimal range of GnT III overexpression in CHO cellsfor ADCC activity, and comparison with oligosaccharide profiles showsthat activity correlates with the level of Fc-associated, bisectedcomplex oligosaccharides.

Given the importance of bisected complex oligosaccharides for ADCCactivity, it would be useful to engineer the pathway to further increasethe proportion of these compounds. Overexpression of GnT III to levelsapproaching that used for sample CE7-30t is within thebiotechnologically practical range where no significant toxicity andgrowth inhibition are observed. At this level of expression, thenon-galactosylated, non-bisected, bi-antennary complex oligosaccharides,i.e., the preferred, potential GnT III substrates, are reduced to lessthan 10% of the total. See, m/z 1486 peak, FIG. 9D. However, only 50%are converted to the desired bisected biantennary complex structures.The rest are either diverted to bisected, hybrid oligosaccharidebyproducts or consumed by the competing enzymeβ1,4-galactosyltransferase, GalT (FIG. 11).

Resolution of the bisected hybrid and the non-bisected, galactosylatedcomplex oligosaccharide peaks by complementary structural analyses woulddetermine how much each potential, undesired route is consuming. Thegrowth of the m/z 1664 and 1810 peaks at high GnT III overexpressionlevels suggests that at least a fraction of these peaks corresponds tobisected hybrid oligosaccharides (FIG. 11). In theory, a flux going tobisected hybrid compounds can be reduced by co-overexpression of enzymesearlier in the pathway such as mannosidase II together with GnT III. Onthe other hand, competition between GnT III and GalT for bisectedcomplex oligosaccharide substrates could potentially be biased towardsGnT III-catalyzed reactions, by increasing the intra-Golgi concentrationof UDP-GlcNAc while overexpressing GnT III. GnT III transfers a GlcNAcfrom the co-substrate UDP-GlcNAc to the different oligosaccharides.Should the intra-Golgi concentration of UDP-GlcNAc co-substrate besub-saturating for GnT III, then increasing it, either by manipulationof the culture medium composition or by genetic manipulation ofsugar-nucleotide transport into the Golgi, could favor GnT III in acompetition for oligosaccharides with GalT.

It remains to be determined whether the increase in ADCC activityresults from the increase in both the galactosylated andnon-galactosylated, bisected complex oligosaccharides, or only from oneof these forms. See, peaks at m/z 1689 and 1851 in FIG. 9. If it isfound that galactosylated, bisected complex bi-antennaryoligosaccharides are the optimal structures for increased ADCC activity,then maximizing the fraction of these compounds on the Fc region wouldrequire overexpression of both GnT III and GalT. Given the competitivescenario discussed previously, the expression levels of both genes wouldhave to be carefully regulated. In addition, it would be valuable to tryto re-distribute overexpressed GalT as much as possible towards the TGNinstead of the trans-Golgi cisterna. The latter strategy may be realizedby exchanging the transmembrane region-encoding sequences of GalT withthose of α2,6-sialyltransferase (Chege and Pfeffer, 1990, J. Cell. Biol.111:893-899).

D. Example 4 Engineering the Glycosylation of the Anti-CD20 MonoclonalAntibody C2B8

C2B8 is an anti-human CD20 chimeric antibody, Reff, M. E. et al, 1994,supra. It received FDA approval in 1997 and is currently being used,under the commercial name of RituxanTM, for the treatment ofNon-Hodgkin's lymphoma in the United States. It is derived from CHO cellculture and therefore should not carry bisected oligosaccharides. See,supra. In order to produce an improved version of this antibody, themethod demonstrated previously for the chCE7 anti-neuroblastoma antibodywas applied. See, supra. C2B8 antibody modified according to thedisclosed method had a higher ADCC activity than the standard,unmodified C2B8 antibody produced under identical cell culture andpurification conditions.

1. Material and Methods

Synthesis Of The Variable Light And Variable Heavy Chain Regions OfChimeric Anti-CD20 MonoclonalAntibody (C2B8). The VH and VL genes of theC2B8 antibody were assembled synthetically using a set of overlappingsingle-stranded oligonucleotides (primers) in a one-step process usingPCR, Kobayashi et al, 1997, Biotechniques 23: 500-503. The sequence datacoding for mouse immunoglobulin light and heavy chain variable regions(VL and VH respectively) of the anti-CD20 antibody were obtained from apublished international patent application (International PublicationNumber: WO 94/11026). The assembled DNA fragments were subcloned intopBluescriptIIKS(+) and sequenced by DNA cycle sequencing to verify thatno mutations had been introduced.

Contruction Of Vectors For Expression Of Chimeric Anti-CD20 MonoclonalAntibody (C2B8). VH and VL coding regions of the C2B8 monoclonalantibody were subcloned in pchCE7H and pchCE7L respectively. In thesubcloning, the sequences coding for the variable heavy and light chainsof the anti-neuroblastoma CE7 (see, supra) were exchanged with thesynthetically assembled variable heavy and variable light chain regionsof C2B8.

Generation Of CHO-tet-GnTIIIm Cells Expressing C2B8 Antibody. The methodfor the generation of a CHO-tet-GntIIIm cell line expressing C2B8antibody was exactly the same as for CHO-tet-GnTIIIm-CE7. See, supra.The clone chosen for further work was named CHO-tet-GnTIIIm-C2B8.

Generation Of CHO-tTA Expressing C2B8 Antibody. CHO-tTA is the parentalcell line of CHO-tet-GnTIIIm. See, supra. The method for the generationof a CHO-tTA cell line expressing C2B8 antibody without GnT IIIexpression was exactly the same as for CHO-tet-GnTIIIm-C2B8 andCHO-tet-GnTIIIm-chCE7. See, supra. The clone chosen for further work wasnamed CHO-tTA-C2B8.

Production Of C2B8 Antibody Samples. Two C2B8 antibody samples werederived from parallel CHO-tet-GnTIIIm-C2B8 cultures; each culturecontaining different levels of tetracycline and therefore expected toexpress GnTIII at different levels. The levels of tetracycline were2000, 50, and 25 ng/ml. The C2B8 antibody samples derived from thesecultures were designated as C2B8-2000t, C2B8-50t, and C2B8-25t,respectively. In parallel, one antibody sample (C2B8-nt) was made from aCHO-tTA-C2B8 culture, this cell line does not express GnT III.CHO-tTA-C2B8 cells were cultured without tetracycline.

Analysis Of GnT III Expression. For Western blot analysis of GnT III,cell lysates of each of the production cultures were resolved bySDS-PAGE and electroblotted to polyvinylidene difluoride membranes.Anti-c-myc monoclonal antibody 9E10 and anti-mouse IgG-horseradishperoxidase (Amersham, Arlington, Ill.) were used as primary andsecondary antibodies respectively. Bound antibody was detected using anenhanced chemiluminiscence kit (Amersham, Arlington, Ill.).

Purification Of C2B8 Antibody Samples. Antibody samples were purifiedusing the same procedure as for the chCE7 antibody samples. See, supra.The concentration was measured using a fluorescence based kit fromMolecular Probes (Leiden, The Netherlands).

Verification Of Specific C2B8 Antigen Binding. The specificity ofantigen binding of the C2B8 anti-CD20 monoclonal antibody was verifiedusing an indirect immunofluorescence assay with cells in suspension. Forthis study, CD20 positive cells (SB cells; ATCC deposit no. ATCC CCL120)and CD20 negative cells (HSB cells; ATCC deposit no. ATCC CCL120.1) wereutilized. Cells of each type were incubated with C2B8 antibody producedat 25 ng/ml tetracycline, as a primary antibody. Negative controlsincluded HBSSB instead of primary antibody. An anti-human IgG Fcspecific, polyclonal, FITC conjugated antibody was used for all samplesas a secondary antibody (SIGMA, St. Louis, Mo.). Cells were examinedusing a Leica (Bensheim, Germany) fluorescence microscope.

ADCC Activity Assay. Lysis of SB cells (CD20+ target cells; ATCC depositno. ATCC CCL120) by human monocyte depleted peripheral blood mononuclearcells (effector cells) in the presence of different concentrations ofC2B8 samples was performed basically following the same proceduredescribed in Brunner et al., 1968, Immunology 14:181-189. The ratio ofeffector cells to target cells was 100:1.

2. Results and Discussion

GnT III Is Expressed At Different Levels In Different Cell Lines AndCultures. The cells of the parallel CHO-tet-GnTIIIm-C2B8 cultures, eachculture containing different levels of tetracycline (2000, 50, and 25ng/ml) and therefore expected to express GnTIII at different levels,were lysed and the cell lysates were resolved by SDS-PAGE and detectedby Western blotting. The lysates of the culture grown at 25 ng/mltetracycline showed an intense band at the corresponding molecularweight of GnT III whereas cultures grown at 50 and at 2000 ng/ml hadmuch less expression of GnT III as shown in FIG. 13.

Verification Of Specific C2B8 Antigen Binding. C2B8 samples producedfrom parallel cultures of cells expressing different levels of GnT IIIwere purified from the culture supernatants by affinity chromatographyand buffer exchanged to PBS on a cation exchange column. Purity wasestimated to be higher than 95% from Coomassie Blue staining of anSDS-PAGE under reducing conditions. These antibody samples were derivedfrom expression of antibody genes whose variable regions weresynthesized by a PCR assembly method. Sequencing of the synthetic cDNAfragments revealed no differences to the original C2B8 variable regionsequences previously published in an international patent application(International Publication Number WO 94/11026). Specific binding of thesamples to human CD20, the target antigen of C2B8, was demonstrated byindirect immunofluorescence using a human lymphoblastoid cell line SBexpressing CD20 on its surface and an HSB lymphoblastoid cell linelacking this antigen. Antibody sample C2B8-25t gave positive staining ofSB cells (FIG. 14A), but not of HSB cells under identical experimentalconditions (see FIG. 14B). An additional negative control consisted ofSB cells incubated with PBS buffer instead of C2B8-25t antibody. Itshowed no staining at all.

In Vitro ADCC Activity Of C2B8 Samples. The antibody sample C2B8-ntexpressed in CHO-tTA-C2B8 cells that do not have Gnt III expression(see, supra) showed 31% cytotoxic activity (at 1 μg/ml antibodyconcentration), measured as in vitro lysis of SB cells (CD20+) by humanlymphocytes (FIG. 15, sample C2B8-nt). C2B8-2000t antibody derived froma CHO-tet-GnTIII culture grown at 2000 ng/ml of tetracycline (i.e., atthe basal level of cloned GnT III expression) showed at 1 μg/ml antibodyconcentration a 33% increase in ADCC activity with respect to theC2B8-nt sample at the same antibody concentration. Reducing theconcentration of tetracycline to 25 ng/ml (sample C2B8-25t), whichsignificantly increased GnTIII expression, produced a large increase ofalmost 80% in the maximal ADCC activity (at 1 μg/ml antibodyconcentration) with respect to the C2B8-nt antibody sample at the sameantibody concentration (FIG. 15, sample C2B8-25t).

Besides exhibiting the highest ADCC activity, C2B8-25t showedsignificant levels of cytotoxicity at very low antibody concentrations.The C2B8-25t sample at 0.06 μg/ml showed an ADCC activity similar to themaximal ADCC activity of C2B8-nt at 1 μg/ml. This result showed thatsample C2B8-25t, at a 16-fold lower antibody concentration, reached thesame ADCC activity as C2B8-nt. This result indicates that the chimericanti-CD20 antibody C2B8 produced in a cell line actively expressing GnTIII was significantly more active than the same antibody produced in acell line that did not express GnT III.

One advantage of this antibody using the methods of the invention isthat (1) lower doses of antibody have to be injected to reach the sametherapeutic effect, having a beneficial impact in the economics ofantibody production, or (2) that using the same dose of antibody abetter therapeutic effect is obtained.

E. Example 5 Establishment of CHO Cell Lines with ConstitutiveExpression of Glycosyltransferase Genes at Optimal Levels Leading toMaximal ADCC Activity

In some applications of the method for enhancing the ADCC it may bedesirable to use constitutive rather than regulated expression of GnTIII on its own or together with other cloned glycosyltransferases and/orglycosidases. However, the inventors have demonstrated that ADCCactivity of the modified antibody depends on the expression level of GnTIII. See, supra. Therefore, it is important to select a clone withconstitutive expression of GnT III alone or together with otherglycosyltransferase and/or glycosidase genes at optimal or near optimallevels. The optimal levels of expression of GnT III, either alone ortogether with other glycosyl transferases such as β(1,4)-galactosyltransferase (GalT), are first determined using cell lines with regulatedexpression of the glycosyl transferases. Stable clones with constitutiveexpression of GnT III and any other cloned glycosyltransferase are thenscreened for expression levels near the optimum.

1. Determination of Near-Optimal Expression Levels Construction of aVector for Regulated GnT III

Expression linked To GFP Expression. Each glycosyl transferase gene islinked, via an IRES sequence, to a reporter gene encoding a proteinretained in the cell, e.g., green fluorescent protein (GFP) or a plasmamembrane protein tagged with a peptide that can be recognized byavailable antibodies. If more than one glycosyl transferase is beingtested, a different marker is associated with each glycosyl transferase,e.g., GnT III may be associated to GFP and GalT may be associated toblue fluorescent protein (BFP). An eucaryotic expression cassetteconsisting of the GnT III cDNA upstream of an IRES element upstream ofthe GFP cDNA is first assembled by standard subcloning and/or PCR steps.This cassette is then subcloned in the tetracycline regulated expressionvector pUHD10-3 (see, supra), downstream of the tet-promoter andupstream of the termination and polyadenylation sequences resulting invector pUHD10-3-GnTIII-GFP.

Establishment Of CHO Cells With Regulated GnTIII Expression Linked ToGFP Expression And Constitutive chCE7 Antibody Expression. CHO-tTA cells(see, supra) expressing the tetracycline-responsive transactivator, areco-transfected with vector pUHD10-3-GnTIII-GFP and vector pPur forexpression of a puromycin-resistance gene. See, supra. Puromycinresistant clones are selected in the presence of tetracycline.Individual clones are cultured by duplicate in the presence (2 μg/ml) orabsence of tetracycline. Six clones that show inhibition of growth inthe absence of tetracycline, due to glycosyltransferase overexpression(see, supra), are selected and analyzed by fluorescence-activated cellsorting (FACS) for detection of the GFP-associated signal. A clonegiving the highest induction ratio, defined as the ratio of fluorescencein the absence of tetracycline to fluorescence in the presence oftetracycline is chosen for further work and designated asCHO-tet-GnTIII-GFP. CHO-tet-GnTIII-GFP are transfected with expressionvectors for antibody chCE7 and a clone with high constitutive expressionof this antibody is selected CHO-tet-GnTIII-GFP-chCE7. See, supra.

Production Of chCE7 Samples, Measurement Of ADCC Activity AndDetermination Of Optimal GnTIII Expression Levels. Parallel cultures ofCHO-tet-GnTIII-GFP-chCE7 are grown at different levels of tetracycline,and therefore expressing GnTIII together with GFP at different levels.chCE7 antibody samples are purified from the culture supernatants byaffinity chromatography. In parallel, the cells from each culture areanalyzed by FACS to determine the mean level of GFP-associatedfluorescence, which is correlated to the expression level of GnT III, ofeach culture. The in vitro ADCC activity of each chCE7 antibody sampleis determined (see, supra) and the maximal in vitro ADCC activity ofeach sample is plotted against the mean fluorescence of the cells usedto produce it.

2. Establishment of a CHO Cell Line with Constitutive GnTIII Expressionat Near-Optimal Levels

The GnTIII-IRES-GFP cassette (see, supra) is subcloned in a constitutiveexpression vector. CHO cells are stably co-transfected with this vectorand a vector for puromycin resistance. Puromycin resistant cells areselected. This population of stably transfected cells is then sorted viaFACS, and clones are selected which express the levels of reporter GFPgene near the within the range where optimal or near-optimal ADCCactivity is achieved. See, supra. This final transfection step may bedone either on CHO cells already stably expressing a therapeuticantibody or on empty CHO cells, e.g., DUKX or DG44 dhfr-CHO cells. Inthe latter case, the clones obtained from the procedure described abovewill be transfected with therapeutic antibody-expression vectors inorder to generate the final antibody-producing cell lines.

F. Example 6 Cell Surface Expression of a Human IgG Fc Chimera withOptimized Glycosylation

Encapsulated cell therapy is currently being tested for a number ofdiseases. An encapsulated cell implant is designed to be surgicallyplaced into the body to deliver a desired therapeutic substance directlywhere it is needed. However, if once implanted the encapsulated devicehas a mechanical failure, cells can escape and become undesirable. Oneway to destroy escaped, undesirable cells in the body is via anFc-mediated cellular cytoxicity mechanism. For this purpose, the cellsto be encapsulated can be previously engineered to express a plasmamembrane-anchored fusion protein made of a type II transmembrane domainthat localizes to the plasma membrane fused to the N-terminus of an Fcregion. Stabila, P. F., 1998, supra. Cells inside the capsule areprotected against Fc-mediated cellular cytoxicity by the capsule, whileescaped cells are accessible for destruction by lymphocytes whichrecognize the surface-displayed Fc regions, i.e., via an Fc-mediatedcellular cytoxicity mechanism. This example illustrates how thisFc-mediated cellular cytoxicity activity is enhanced by glycosylationengineering of the displayed Fc regions.

1. Establishment of Cells Expressing the Fc Chimera on their Surface andExpressing GnTIII

Cells to be implanted for a particular therapy, for example baby hamsterkidney (BHK) cells, which already produce the surface-displayed Fcchimera and a secreted, therapeutic protein, are first stablytransfected with a vector for constitutive expression of GnTIII linkedvia an IRES element to expression of GFP. See, supra. Stabletransfectants are selected by means of a marker incorporated in thevector, e.g., by means of a drug resistance marker and selected forsurvival in the presence of the drug.

2. Screening of Cells Expressing Diffent Levels of GnTIII andMeasurement

Stable transfectants are analyzed by fluorescence-activated cell sorting(FACS) and a series of clones with different mean fluorescence levelsare selected for further studies. Each selected clone is grown andreanalyzed by FACS to ensure stability of GFP, and therefore associatedGnT III, expression.

3. Verification of Different Levels of Bisected Complex Oligosaccharideson the Displayed Fc Regions

Fc regions from three clones with different levels of GFP-associatedfluorescence and from the original BHK cells not transfected with theGnTIII-IRES-GFP vector are solubilized from the membrane by means of adetergent and then purified by affinity chromatography. Theoligosaccharides are then removed, purified and analyzed byMALDI-TOF/MS. See, supra. The resulting MALDI-TOF/MS profiles show thatthe Fc-regions of the modified, fluorescent clones carry differentproportions of bisected complex oligosaccharides. The MALDI profile fromthe unmodified cells does not show any peak associated to bisectedoligosaccharides. The clone with carrying the highest levels of bisectedcomplex oligosaccharides on the displayed Fc regions is chosen forfurther work.

4. In vitro Fc-mediated Cellular Cytoxicity Activity Assay

Two Fc-mediated cellular cytoxicity activity assays are then conductedin parallel. In one assay the target cells are derived from the cloneselected above. In the parallel assay the target cells are the originalcells to be encapsulated and which have not been modified to expressGnTIII. The assay is conducted using the procedure described previously(see, supra) but in the absence of any additional antibody, since thetarget cells already display Fc regions. This experiment demonstratesthat the Fc-mediated cellular cytoxicity activity against the cellsexpressing GnT III is higher than that against cells not expressing thisglycosyltransferase.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

1. A glycoengineered Chinese Hamster Ovary cell that expresses arecombinant antibody comprising an IgG Fc region or fragment thereofcontaining N-linked oligosaccharides, wherein said Chinese Hamster Ovarycell has been genetically manipulated to have altered activity of atleast one glycoprotein-modifying glycosyltransferase, wherein saidantibody has an altered pattern of glycosylation in the Fc regioncompared to the corresponding antibody produced by the same ChineseHamster Ovary cell that has not been glycoengineered, and wherein saidantibody has increased Fc-mediated cellular cytotoxicity as a result ofsaid altered glycosylation.
 2. A glycoengineered Chinese Hamster Ovarycell that expresses a recombinant antibody comprising an IgG Fc regionor fragment thereof containing N-linked oligosaccharides, wherein saidChinese Hamster Ovary cell has been genetically manipulated to havealtered activity of at least one glycoprotein-modifyingglycosyltransferase, wherein said antibody has an altered pattern ofglycosylation in the Fc region compared to the corresponding antibodyproduced by the same Chinese Hamster Ovary cell that has not beenglycoengineered, and wherein said antibody has increased Fc receptorbinding affinity as a result of said altered glycosylation.
 3. Aglycoengineered Chinese Hamster Ovary cell according to claim 1 or claim2, wherein said altered pattern of glycosylation comprises an increasedproportion of nonfucosylated oligosaccharides.
 4. A glycoengineeredChinese Hamster Ovary cell according to claim 1 or claim 2, wherein thepredominant N-linked oligosaccharide in the Fc region of said antibodyis nonfucosylated.
 5. A glycoengineered Chinese Hamster Ovary cellaccording to claim 1 or claim 2, wherein said antibody is a chimericantibody.
 6. A glycoengineered Chinese Hamster Ovary cell according toclaim 1 or claim 2, wherein said antibody is a humanized antibody.
 7. Aglycoengineered Chinese Hamster Ovary cell according to claim 1 or claim2, wherein said antibody is a fusion protein that includes a Fc regionof an immunoglobulin.
 8. A glycoengineered Chinese Hamster Ovary cellaccording to claim 1 or claim 2, wherein the predominant N-linkedoligosaccharide in the Fc region is not a high-mannose structure.
 9. Aglycoengineered Chinese Hamster Ovary cell according to claim 3, whereinsaid Fc region containing N-linked oligosaccharides further comprises anincreased proportion of GlcNAc residues compared to the correspondingantibody produced by the same Chinese Hamster Ovary cell that has notbeen glycoengineered.
 10. A glycoengineered Chinese Hamster Ovary cellthat produces a recombinant antibody comprising an Fc region containingN-linked oligosaccharides, wherein said Chinese Hamster Ovary cell hasbeen genetically manipulated to have altered activity of at least oneglycoprotein-modifying glycosyltransferase, wherein said antibody has anincreased proportion of GlcNAc residues in the Fc region relative to theproportion of fucose residues compared to the proportion of GlcNAc tofucose residues in the Fc region of a corresponding antibody produced bythe same Chinese Hamster Ovary cell that has not been glycoengineered,and wherein said antibody has increased Fc-mediated cellularcytotoxicity as a result of said genetic manipulation.
 11. Aglycoengineered Chinese Hamster Ovary cell according to claim 10,wherein said GlcNAc residues are bisecting.
 12. A glycoengineeredChinese Hamster Ovary cell according to claim 10, wherein said GlcNAcresidues are bisecting and wherein said bisected oligosaccharides are ofcomplex type.
 13. A glycoengineered Chinese Hamster Ovary cell accordingto claim 10, wherein said GlcNAc residues are bisecting and wherein saidbisected oligosaccharides are of hybrid type.
 14. A glycoengineeredChinese Hamster Ovary cell according to claim 10, wherein said at leastone glycoprotein-modifying glycosyltransferase is selected from thegroup consisting of: β(1,4)-N-acetylglucosaminyltransferase III,β(1,4)-N-acetylglucosaminyltransferase V, β(1,4) -galactosyltransferase,α-mannosidase II, and core α-1,6-fucosyltransferase.
 15. Aglycoengineered Chinese Hamster Ovary cell according to claim 14,wherein said at least one glycoprotein-modifying glycosyltransferase isβ(1,4)-N-acetylglucosaminyltransferase III.
 16. A glycoengineeredChinese Hamster Ovary cell according to claim 14, wherein said at leastone glycoprotein-modifying glycosyltransferase is coreα-1,6-fucosyltransferase.
 17. A glycoengineered Chinese Hamster Ovarycell according to claim 14, wherein said at least oneglycoprotein-modifying glycosyltransferase is α-mannosidase II.
 18. Aglycoengineered Chinese Hamster Ovary cell according to claim 14,wherein said altered activity is increased activity of said at least oneglycoprotein-modifying glycosyltransferase.
 19. A glycoengineeredChinese Hamster Ovary cell according to claim 14, wherein said alteredactivity is decreased activity of said at least oneglycoprotein-modifying glycosyltransferase.
 20. A glycoengineeredChinese Hamster Ovary cell according to claim 18, wherein said at leastone glycoprotein-modifying glycosyltransferase isβ(1,4)-N-acetylglucosaminyltransferase III.
 21. A glycoengineeredChinese Hamster Ovary cell according to claim 20, wherein said increasedactivity is increased expression ofβ(1,4)-N-acetylglucosaminyltransferase III.
 22. A glycoengineeredChinese Hamster Ovary cell according to claim 19, wherein said at leastone glycoprotein-modifying glycosyltransferase is coreα1,6-fucosyltransferase.
 23. A glycoengineered Chinese Hamster Ovarycell according to claim 17, wherein said altered activity is increasedexpression of β(1,4)-N-acetylglucosaminyltransferase III andα-mannosidase II.
 24. A glycoengineered Chinese Hamster Ovary cellaccording to claim 17, wherein said altered activity is increasedexpression of β(1,4)-N-acetylglucosaminyltransferase III andα-mannosidase II and β(1,4)-galactosyltransferase.
 25. A glycoengineeredChinese Hamster Ovary cell according to claim 1 or claim 2, wherein saidantibody is a therapeutic antibody.
 26. A glycoengineered ChineseHamster Ovary cell according to claim 1 or claim 2, wherein saidantibody selectively binds to an antigen expressed by cancer cells. 27.A glycoengineered Chinese Hamster Ovary cell according to claim 25,wherein said antibody is a monoclonal antibody.
 28. A glycoengineeredChinese Hamster Ovary cell according to claim 25, wherein said antibodyis a selected from the group consisting of: an anti-CD20 antibody, ananti-human neuroblastoma antibody, an anti-human renal cell carcinomaantibody, an anti-HER2 antibody, an anti-human colon, lung, and breastcarcinoma antibody, an anti-human 17-1A antigen antibody, a humanizedanti-human colorectal tumor antibody, an anti-human melanoma antibody,and an anti-human squamous-cell carcinoma antibody.
 29. Aglycoengineered Chinese Hamster Ovary cell according to claim 1 or claim2, wherein the majority of the N-linked oligosaccharides in the Fcregion of said antibody are bisected.
 30. A glycoengineered ChineseHamster Ovary cell according to claim 1 or claim 2, wherein the majorityof the N-linked oligosaccharides in the Fc region of said antibody arenonfucosylated.
 31. A glycoengineered Chinese Hamster Ovary cellaccording to claim 1 or claim 2, wherein the majority of the N-linkedoligosaccharides in said Fc region of said antibody are bisected,nonfucosylated.
 32. A glycoengineered Chinese Hamster Ovary cellaccording to claim 1 or claim 2, wherein said Chinese Hamster Ovary cellis produced by a process comprising: (a) providing a Chinese HamsterOvary cell comprising at least one nucleic acid encoding a recombinantantibody; and (b) genetically manipulating said Chinese Hamster Ovarycell to alter the activity in said cell of at least oneglycoprotein-modifying glycosyltransferase.
 33. A glycoengineeredChinese Hamster Ovary cell according to claim 32, wherein said geneticmanipulation is achieved by introducing into said cell at least one geneencoding an exogenous glycoprotein-modifying glycosyltransferase.
 34. Aglycoengineered Chinese Hamster Ovary cell according to claim 25,wherein said antibody is a therapeutic monoclonal antibody having ahuman Fc region and that selectively binds an antigen expressed bycancer cells, and wherein the majority of oligosaccharides in the Fcregion of said antibody are nonfucosylated.
 35. A glycoengineeredChinese Hamster Ovary cell according to claim 1 or claim 2, wherein atleast 45% of the oligosaccharides in the Fc region are complexstructures.
 36. A glycoengineered Chinese Hamster Ovary cell accordingto claim 1 or claim 2, wherein said recombinant antibody exhibits atleast an 80% increase in maximal ADCC activity compared to the sameantibody produced by the same Chinese Hamster Ovary cell under identicalculture and purification conditions, but which has not beenglycoengineered.
 37. A glycoengineered Chinese Hamster Ovary cellaccording to claim 1 or claim 2, wherein said at least oneglycoprotein-modifying glycosyl transferase is mammalian.
 38. Aglycoengineered Chinese Hamster Ovary cell according to claim 37,wherein said at least one glycoprotein-modifying glycosyl transferase ishuman.
 39. A glycoengineered Chinese Hamster Ovary cell according toclaim 33, wherein said exogenous glycoprotein-modifyingglycosyltransferase is β(1,4)-N-acetylglucosaminyltransferase III andα-mannosidase II.
 40. A glycoengineered Chinese Hamster Ovary cellaccording to claim 33, wherein said exogenous glycoprotein-modifyingglycosyltransferase is α-mannosidase II.
 41. A glycoengineered ChineseHamster Ovary cell according to claim 1 or claim 2, wherein said IgG Fcregion containing N-linked oligosaccharides comprises an entire IgG Fcregion.
 42. A glycoengineered Chinese Hamster Ovary cell according toclaim 1 or claim 2, wherein said IgG Fc region containing N-linkedoligosaccharides comprises an IgG fragment.
 43. A glycoengineeredChinese Hamster Ovary cell according to claim 42, wherein said IgGfragment comprises a CH2 domain.